Transgenic plants expressing maize acetyl COA carboxylase gene and method of altering oil content

ABSTRACT

The present invention provides a complete cDNA sequence and partial DNA sequences encoding maize acetyl CoA carboxylase and methods for altering the oil content of plants by introducing and expressing a maize acetyl CoA carboxylase gene in plant cells.

This application is a national stage filing of PCT/US96/04625, filed Apr. 4, 1996, which is a continuation-in-part of U.S. application Ser. No. 08/417,089, filed Apr. 5, 1995, now U.S. Pat. No. 6,069,298 which is a continuation-in-part of U.S. application Ser. No. 08/014,326, filed Feb. 5, 1993, now U.S. Pat. No. 5,498,544.

BACKGROUND OF THE INVENTION

Acetyl CoA carboxylase (ACCase) is an enzyme involved in many important metabolic pathways in plant, animal and bacterial cells. The enzyme is especially important in fatty acid synthesis in plants and is sensitive to inhibition by some types of herbicides. Structurally, ACCases are biotinylated and are quite large enzymes consisting of one or more subunits. For example, most ACCases of animals, higher plants, and yeast are dimers of 420 to 700 kD native MW and contain subunits of 200 to 280 kD. Diatom and algal ACCases are 700 to 740 kD tetramers of 160 to 180 kD subunits. Bacterial ACCase consists of three dissociable proteins, biotin carboxylase (51 kD), biotin carboxyl carrier protein (22.5 kD), and biotin transcarboxylase (130 kD).

Acetyl CoA Carboxylase (ACCase) catalyzes the formation of malonyl-CoA from acetyl-CoA and bicarbonate in animal, plant, and bacterial cells. Malonyl-CoA is an essential substrate for (i) de novo fatty acid (FA) synthesis, (ii) fatty acid elongation, (iii) synthesis of secondary metabolites such as flavonoids and anthocyanins, and (iv) malonylation of some amino acids and secondary metabolites. Synthesis of malonyl-CoA is the first committed step of flavonoid and fatty acid synthesis and current evidence suggests that ACCase catalyzes the rate-limiting step of fatty acid synthesis. Formation of malonyl-CoA by ACCase occurs via two partial reactions and requires a biotin prosthetic group:

(i)E-biotin+ATP+HCO₃→E-biotin-CO₂+ADP+Pi

(ii)E-biotin-CO₂+Acetyl-CoA —>E-biotin+malonyl-CoA (NET)Acetyl-CoA+ATP+HCO₂→malonyl-CoA+ADP+Pi

In E. coli, these reactions are catalyzed by three distinct components; biotin carboxylase, biotin transcarboxylase, and biotin carboxyl carrier protein, which can be separated and yet retain partial activity. Plant and animal ACCases contain all three activities on a single polypeptide.

In plants, most ACCase activity is located in plastids of green and non-green plant tissues including leaves and oil seeds. Leaf ACCase activity is primarily located in mesophyll cells, but lesser amounts have been found in C-4 bundle sheath cells and in epidermal cells. The subcellular location of ACCase activity in epidermal cells is unknown, but since synthesis of very long-chain fatty acids (VLCFA) for formation of waxes, cutin, and suberin occurs on the endoplasmic reticulum (ER), malonyl-CoA might also be derived from a cytosolic ACCase. In contrast, rat ACCase is primarily cytosolic or associated with the outer mitochondrial membrane.

De novo fatty acid synthesis in chloroplasts involves successive 2-carbon additions to acetate, using malonate as the 2-C donor. All intermediates are attached to acyl carrier protein (ACP). Synthesis in plastids resembles that in E. coli in that the fatty acid synthesis complex can be dissociated into separate enzymes: β-ketoacyl-ACP synthase (KAS), P-ketoacyl-ACP reductase, β-hydroxyl-ACP dehydratase, and enoyl-ACP reductase, acetyl-CoA:ACP transacylase, and malonyl-CoA:ACP transacylase. A highly active KASIII isozyme catalyzes the condensation of acetyl-CoA and malonyl-ACP. Successive additions of malonyl-CoA to acy-1-ACPs catalyzed by KAS I form C16 acyl-ACP, some of which is converted to C18 acyl-ACP by KAS II and then to C18:1-ACP. Fatty acid inetabolisni then diverges; de-esterification allows movement to the cytoplasm (eukaryootic path) where fatty acids may be further unsaturated and/or elongated by additions of malonyl-CoA in the ER. Alternatively, fatty acids are linked to glycerol-3-phosphate (prokaryotic path), further unsaturated, and used for synthesis of chloroplast lipids. A portion of cytoplasmic lipids returns to the chloroplast. The relative contributions of these two paths are species-specific but appear to be relatively flexible in mutants blocked in either path. In oil-storing organs such as cotyledons and monocot embryos the triacylglycerides are stored in cytoplasmic oil bodies surrounded by a single unit membrane.

Condensation of malonyl-CoA with phenylpropionyl-CoAs or acetyl-CoA leads to synthesis of flavonoids, anthocyanins, or to polyacetates. Condensation is increased by light, elicitors, or pathogens and may be the rate-limiting step in synthesis of some phytoalexins. In addition to the secondary metabolites derived by de novo synthesis, malonyl conjugates of flavonoid glycosides, formed by malonyl-CoA:flavonoid glycoside malonyltransferase, D-amino acids and 1-amino-carboxyl-cyclopropane (ethylene precursor) are found in plants. Malonylated compounds accumulate in vacuoles, probably after synthesis in the cytoplasm.

An important property of ACCase is the central role it plays in fatty acid synthesis and accumulation in plants and seeds. Available evidence supports the idea that ACCase activity is the rate-limiting step for de novo fatty acid synthesis in plants. High rates of ACCase activity in vitro parallel or slightly precede high rates of lipid deposition or [¹⁴C] acetate incorporation into lipids in developing leaves and oil seeds. Significant changes in plant ACCase activity occur during chloroplast development and increase in ACCase activity correlates with lipid deposition in developing oil seeds. Turnham et al., Biochem. J. 212:223 (1883); and Beittenmiller et al., Plant Physiol., 100:923 (1992).

Among other properties, ACCase in most monocots is also inhibited by several herbicides. [¹⁴C]acetate incorporation into maize lipids is strongly inhibited by fluazifop and sethoxydim due to inhibition of plastid ACCase. In barley however, fluazifop had little effect on [¹⁴C]acetate incorporation into very long-chain fatty acids. Since synthesis of very long-chain fatty acids occurs in the cytosol on the ER, and de novo fatty acid synthesis occurs in the plastids, cytosolic malonyl-CoA might be supplied by a herbicide insensitive ACCase isozyme.

There are three general mechanisms by which plants may be resistant to, or tolerant of, herbicides. These mechanisms include insensitivity at the site of action of the herbicide (usually an enzyme), rapid metabolism (conjugation or degradation) of the herbicide, or poor uptake and translocation of the herbicide. Altering the herbicide site of action from a sensitive to an insensitive form is the preferred method of conferring tolerance on a sensitive plant species. This is because tolerance of this nature is likely to be a dominant trait encoded by a single gene, and is likely to encompass whole families of compounds that share a single site of action, not just individual chemicals.

Therefore, detailed information concerning the biochemical site and mechanism of herbicide action is of great importance and can be applied in two ways. First, the information can be used to develop cell selection strategies for the efficient identification and isolation of appropriate herbicide-tolerant variants. Second, it can be used to characterize the variant cell lines and regenerated plants that result from the selections.

Tissue culture methods have been used to select for resistance (or tolerance) using a variety of herbicides and plant species (see review by Meredith and Carlson, 1982, in Herbicide Resistance in Plants, eds. Lebaron and Gressel, pp. 275-291, John Wiley and Sons, NY). For example, P. C. Anderson et al., in U.S. Pat. No. 4,761,373, disclose the use of tissue culture methods to produce maize plants resistant to herbicidal imidazolidones and sulfonamides.

The resistance is due to the presence of altered acetohydroxy acid synthase which is resistant to deactivation by these herbicides.

Certain 1,3-cyclohexanediones exhibit general and selective herbicidal activity against plants. One such cyclohexanedione is sethoxydim {2-[1-(ethoxyimino)-butyl]-5-[2-(ethylthio)propyl]-3-hydroxy-2-cyclohexen-1-one}. Sethoxydim is commercially available from BASF (Parsippany, N.J.) under the designation POAST™.

Other herbicidal cyclohexanediones include clethodim, (E,E)-(±)-2-[1-[[(3-chloro-2-propenyl)oxy]imino]propyl]-5-[2-(ethylthio)propyl]-3-hydroxy-2-cyclohexen-1-one; available as SELECT™ from Chevron Chemical (Valent) (Fresno, Calif.); cloproxydim, (E,E)-2-[1-[[(3-chloro-2-propenyl)oxy]imino]butyl]-5-[2-(ethylthio)propyl]-3-hydroxy-2-cyclohexen-1-one: available as SELECTONE™ from Chevron Chemical (Valent) (Fresno, Calif.); and tralkoxydim, 2-[1-(ethoxyimino)propyl]-3-hydroxy-5-mesitylcyclohex-2-enone, available as GRASP™ from Dow Chemical USA (Midland. Mich.).

For purposes of reference in the present specification, the herbicides described in the two preceding paragraphs and other structurally related herbicidal compounds, are collectively referred to as the cyclohexanedione family of herbicides.

Certain aryloxyphenoxypropanoic acids exhibit general and selective herbicidal activity against plants. In these compounds, the aryloxy group may be phenoxy, pyridinyloxy or quinoxalinyl. One such herbicidal aryloxyphenoxypropanoic acid is haloxyfop, {2-[4-[[3-chloro-5-(trifluoromethyl)-2-pyridinyl]oxy]phenoxy]-propanoic acid}, which is available as VERDICT™ from Dow Chemical USA (Midland, Mich.). Another is diclofop, {(±)-2-[4-(2,4-dichlorophenoxy)-phenoxy]propanoic acid}, available as HOELON™ from Hoechst-Roussel Agri-Vet Company (Somerville. N.J).

Other members of this family of herbicides include fenoxyaprop, (±)-2-[4-[(6-chloro-2-benzoxazolyl)oxy]phenoxy]propanoic acid; available as WHIP™ from Hoechst-Roussel Agri-Vet Company (Somerville, N.J.); fluazifop, (±)-2-[4-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenoxy]propanoic acid; available as FUSILADE™ from ICI Americas (Wilmington. Del.); fluazifop-P, (R)-2-[4-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenoxy]propanoic acid; available as FUSILADE 2000™ from ICI Americas (Wilmington. Del.): and quizalofop, (±)-2-[4[(6-chloro-2-quinoxalinyl)oxy]phenoxy]propanoic acid; available as ASSURE™ from E. I. DuPont de Nemours (Wilmington, Del.).

For purposes of reference in the present specification, the herbicides referred to in the two preceding paragraphs and other structurally related herbicidal compounds, are collectively referred to as herbicidal aryloxyphenoxypropanoic acids.

Thus, there is a need for methods to develop plants that are resistant or tolerant to herbicides. There is also a need to increase the oil and/or fatty acid content of the plants and seeds, as well as for methods to increase their resistance to herbicides. Moreover, there is a need to identify and clone genes important in conferring herbicide tolerance and in increasing the oil content of plants.

SUMMARY OF THE INVENTION

The present invention provides an isolated and purified DNA molecule comprising a DNA segment encoding a maize acetyl CoA carboxylase gene and methods for conferring herbicide tolerance and/or altering the oil content of plants by introducing and expressing a plant acetyl CoA carboxylase gene in the plant cells. The DNA molecule encoding a plant acetyl CoA carboxylase can encode an unaltered plant acetyl CoA carboxylase or an altered plant acetyl CoA carboxylase substantially tolerant to inhibition by cyclohexanedione or aryloxyphenoxypropanoic acid herbicides as well as encoding an antisense DNA sequence that is substantially complementary to a plant acetyl CoA carboxylase gene or to a portion thereof. A DNA molecule of the invention can also further comprise an amino terminal plant chloroplast transit peptide sequence operably linked to the maize acetyl CoA carboxylase gene.

The method of imparting cyclohexanedione or aryloxyphenoxypropanoic acid herbicide tolerance to a plant includes the steps of introducing a chimeric DNA molecule comprising a gene coding for a plant acetyl CoA carboxylase or an altered or a functional mutant thereof operably linked to a promoter functional in a plant cell into cells of a susceptible plant, and regenerating the transformed plant cells to provide a differentiated plant. The promoter can be an inducible or tissue specific promoter or provide for overexpression of at least about a 2-fold amount of a native plant acetyl CoA carboxylase. The functional linkage of a promoter to the chimeric DNA molecule results in an expression cassette. Expression of the chimeric DNA molecule is in an amount effective to render the acetyl CoA carboxylase and/or the plant tissue substantially tolerant to the herbicides relative to the native acetyl CoA carboxylase present in said plant. Herbicide tolerance can be achieved in the plants by at least two methods, including increasing the level of gene expression of a native or unaltered acetyl CoA carboxylase, or by introducing an altered gene coding for an acetyl CoA carboxylase that is less sensitive to herbicide inhibition. The level of gene expression can be increased by either combining a plant acetyl CoA carboxylase gene with a promoter that provides for a high level of gene expression, such as a 35S cauliflower mosaic virus promoter (CaMV), or by introducing multiple copies of the gene into the cell so that the multiple copies of the gene are integrated into the genome of transformed plant cells. The preferred plant cells into which to introduce the expression cassette of the invention, to achieve herbicide tolerance, are monocot plant cells. Once transformed cells exhibiting herbicide tolerance are obtained, transgenic plants and seeds can then be regenerated therefrom, and evaluated for stability of the inheritance of the herbicide tolerance trait.

The invention also provides a method for altering, preferably raising, the oil content in a plant. The method includes the steps of introducing a chimeric DNA molecule comprising a gene coding for a plant acetyl CoA carboxylase or an altered or a functional mutant thereof operably linked to a promoter functional in a plant cell into the cells of plant tissue and expressing the gene in an amount effective to alter the oil content of the plant cell. An alteration in oil content can include a change in total oil content over that normally present in that type of plant cell or a change in the type of oil present in the cell. An alteration in oil content in the plant cell, according to the method of the invention can be achieved by at least two methods including:

(1) an increase or decrease in expression of an altered plant acetyl CoA carboxylase gene; or

(2) by introducing an altered or functional mutant plant acetyl CoA carboxylase gene.

The level of gene expression of an unaltered plant acetyl CoA carboxylase gene can be increased by either combining an unaltered plant acetyl CoA carboxylase with a promoter that provides for a high level of gene expression, or by introducing multiple copies of an expression cassette into cells so that multiple copies of the gene are integrated into the genome. When an altered or a functional mutant plant acetyl CoA carboxylase gene codes for an enzyme that exhibits an increase in specific activity, it can lead to an increase in total oil content of the plant cell. When an altered or a functional mutant acetyl CoA carboxylase gene codes for an enzyme having a decrease in specific activity, it may lead to a decrease in the total oil content of the plant cell. Preferably, the expression cassette is introduced into dicot plants such as soybeans, canola, and sunflower. In an especially preferred version, transformed cells exhibiting about a 1.2- to 5-fold increase in total oil content and/or expression or specific activity of acetyl CoA carboxylase are selected for and used to generate transgenic plants and seeds exhibiting a substantial increase in oil content. A substantial increase in oil content depends on the oil content normally present in the plant or seed and can range from about a 1.2 to a 20-fold increase.

The invention also provides for a method of producing plant acetyl CoA carboxylase in a host cell. The method includes the steps of introducing an expression cassette comprising a chimeric gene encoding a plant acetyl CoA carboxylase or an altered or a functional plant acetyl CoA carboxylase operably linked to a promoter into a host cell and expressing the gene in an amount sufficient to permit crystallization of the plant acetyl CoA carboxylase. An expression cassette can include a promoter that is functional in either a eukaryotic or a prokaryotic cell. Preferably, the expression cassette is introduced into a prokaryotic cell, such as E. coli, that is routinely used for production of recombinantly produced proteins. Recombinantly produced and crystallized plant acetyl CoA carboxylase can then be used to identify other herbicides and that bind to and inhibit acetyl CoA carboxylase in plants. In addition, the availability of large amounts of purified enzyme can permit the screening of the efficacy of such herbicides in terms of their ability to bind to, or otherwise inhibit, the activity of the enzyme.

The present invention also provides an isolated and purified DNA molecule of at least seven nucleotide bases which hybridizes under high stringency conditions to a DNA molecule comprising) a DNA segment encoding a plant acetyl CoA carboxylase and provides a hybridization probe comprising an isolated and purified DNA molecule of at least seven nucleotide bases, which is detectably labeled or which binds to a detectable label, which DNA molecule hybridizes under high stringency conditions to the non-coding strand of a DNA molecule comprising a DNA segment encoding a plant acetyl CoA carboxylase.

High stringency conditions are defined as: hybridization at 65° C. for at least 16 hours in 5×SSC, 1×Denhardt's solution, 50 mM Tris-HCl, pH 8, 0.2% SDS, 10 mM EDTA, 0.1 mg/ml salmon sperm DNA, followed by washing twice for 5 minutes in 2×SSC, 0.5% SDS at 25° C., once for 10 minutes in 0.2×SSC, 0. 1% SDS at 25° C. and twice for 30 minutes in 0.2×SSC, 0.1% SDS at 65° C.

The present invention also provides a method of introducing an exogenous plant acetyl CoA carboxylase gene into a host cell comprising transforming host cells in vitro with an expression cassette comprising a chimeric DNA molecule encoding a plant acetyl CoA carboxylase gene operably linked to a promoter functional in the host cell, expanding the transformed host cells in vitro, and identifying a transformed host cell which expresses the chimeric DNA molecule.

The term “consists essentially of” as used with respect to the present DNA molecules is defined to mean that a major portion of the nucleotide sequence encodes an ACCase, and that nucleotide sequences are not present which encode proteins other than ACCase or functional equivalents thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic depiction of the fatty acid biosynthesis pathway in plants.

FIG. 2 is a graph depicting the effect of sethoxydim on the growth of mutant maize callus.

FIG. 3 is a graph depicting the shoot length growth of maize seedlings seven days after treatment with sethoxydim.

FIG. 4 is a graph depicting the shoot length growth of maize seedlings fourteen days after treatment with sethoxydim.

FIG. 5: Total soluble and biotinylated polypeptides in ACCase purification fractions from seedling leaves of maize inbred A619. Proteins were separated by SDS-PAGE in 7.5% gels and then silver-stained (Panel A). An identical gel was Western-blotted and a longitudinal section of each lane was probed with avidin (Panel B). Lanes were 1: crude (10 μg); 2: (NH₄)₂SO₄ (10 μg); 3: S-300 (5 μg); 4: Blue Sepharose (2 μg); 5: Mono-Q ACCase II (5 μg); and 6: Mono-Q ACCase I (5 μg). Diagonal lines between lanes indicate position of molecular weight markers shown on the left.

FIG. 6: Immunoprecipitation of ACCase activity from B73 leaf, embryo, endosperm, and BMS suspension cultured cells. Equal activities (0.58 nmol min⁻¹ ) were incubated with 16 μL serum (immune plus preimmune), immune complexes were precipitated with Protein A-agarose, and ACCase activity remaining in the resulting supernatant fraction was measured relative to the preimmune serum control.

FIG. 7: Herbicide inhibition of acetyl-(AcCoA) or propionyl-CoA (Prop-CoA)-dependent H¹⁴CO₃-incorporation into acid-stable product by ACCase I and II Mono-Q fractions. Activities in the presence of haloxyfop (1 μM) are expressed relative to the minus herbicide control.

FIG. 8: Comparison of the peptide sequence of maize cDNA clones #15-14 and #18-5 with chicken ACCase. The approximate locations of the biotin carboxylase, biotin binding site, and biotin transcarboxylase functional domains are indicated for the chicken sequence. The percentages of amino acid identity are indicated by cross-hatched boxes for the maize coding sequence. Regions of genomic DNA Type I and Type 11 clone sequences that align with cDNA #18-5 are indicated by solid heavy lines. The approximate locations of subclone #28 and #16 from genomic Type I and subclone #34 from genomic Type II clones are indicated.

FIG. 9: Northern blot of total RNA from leaf, immature embryo and endosperm tissue (16 days after pollination), and Black Mexican Sweet corn (BMS) cells. Lanes contain 10 μg total RNA and were probed with the 2 kb EcoRI fragment of lambda clone #15-14.

FIGS. 10A-10B: DNA sequence (SEQ ID NO. I) of a 2 kb EcoRI fragment of lambda clone #15-14 including a portion of a maize ACCase gene located at bases 2883 to 83 from the 3′ stop codon.

FIG. 11: Graph of ACCase activity during seed development in two high oil soybean cell lines and one low oil soybean cell line.

FIG. 12: Cloning strategy to obtain the complete coding sequence of the maize ACCase gene.

FIGS. 13A-13E: DNA sequence (SEQ ID NO:5) of a 7470 base pair cDNA of a maize ACCase gene. (Genbank Accession No. U19183).

FIGS. 14A-14B: Predicted amino acid sequence of the complete ACCase gene of maize (SEQ ID NO:6).

FIG. 15: Restriction map of ACCase genomic clones.

FIGS. 16A-16B: Partial nucleotide sequence of a Type A1 ACCase genomic clone (SEQ ID NO:12).

FIG. 17: Partial nucleotide sequence of clone 5A, a Type A ACCase genomic clone (SEQ ID NO:13).

FIGS. 18A-18F: Partial nucleotide sequence of six Type A, ACCase genomic clones (SEQ ID NOs 14,15, 16. 17, 18. and 19).

FIGS. 19A-19C: Partial nucleotide sequence of three Type B ACCase clones (SEQ ID NOs 20, 21 and 22).

FIGS. 20A-20B: SDS/PAGE analysis of chloroplast importation of ³⁵S-labeled ACCase polypeptides. (A) Pea and maize chloroplasts incubated with ³⁵5-labeled ACCase polypeptides for 30 minutes. (B) A time course analysis of the importation of ³⁷S-labeled ACCase polypeptides into maize chloroplasts.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a DNA molecule encoding a plant acetyl CoA carboxylase gene and methods for conferring herbicide tolerance and/or altering the oil content of plants by introducing and expressing a plant acetyl CoA carboxylase gene in the plant cells. In plants, acetyl CoA carboxylase plays a central role in regulating fatty acid synthesis and in the sensitivity of monocots to cyclohexanedione or aryloxyphenoxypropanoic acid herbicides.

In accord with the present invention, a plant acetyl CoA carboxylase gene is identified, isolated and combined with a promoter functional in a plant cell to provide a recombinant expression cassette. A plant acetyl CoA carboxylase gene can be introduced and expressed in a plant cell. Depending on the type of plant cell, the level of gene expression, and the activity of the enzyme encoded by the gene, introduction of a plant acetyl CoA carboxylase gene into the plant cell can confer herbicide tolerance and/or alteration of the oil of the plant cell.

In monocots, an exogenously introduced plant acetyl CoA carboxylase gene can be expressed at a level effective to render the cells of the plant tissue substantially tolerant to cyclohexanedione or aryloxyphenoxypropanoic acid herbicide levels which normally inhibit a native or endogenous acetyl CoA carboxylase. A native acetyl CoA carboxylase i-i an enzyme that is normally encoded and expressed in the plant cell prior to transformation. An exogenously introduced plant acetyl CoA carboxylase gene is a gene which has been isolated and amplified from either the same or different type of cell. Exogenous introduction and expression of a plant acetyl CoA carboxylase gene in both monocots and dicots can result in alteration of the oil content and quality of plant tissue and seeds. Exogenous introduction and expression in a host cell, such as a bacteria, can provide for sufficient amounts of plant acetyl CoA carboxylase to allow for crystallization and isolation of the enzyme. Crystallized plant acetyl CoA carboxylase is useful to identify other herbicides that bind to and can inhibit plant acetyl CoA carboxylases. The enzyme could also be used to screen potential herbicidal compounds for efficacy.

A. Formation of an Expression Cassette

An expression cassette of the invention can comprise a chimeric DNA molecule encoding a plant acetyl CoA carboxylase gene or an altered or functional mutant thereof operably linked to a promoter functional in a plant cell. The gene can code for a plant acetyl CoA carboxylase that is substantially tolerant to herbicides, preferably cyclohexanedione and/or aryloxyphenoxypropanoic acid herbicides. An expression cassette of the invention can also include an antisense DNA sequence that is substantially complementary to an acetyl CoA carboxylase gene or a portion thereof operably linked to a promoter functional in a plant cell.

1. Isolation and Identification of a Gene Coding for a Plant Acetyl CoA Carboxylase

A gene encoding a plant acetyl CoA carboxylase can be identified and isolated by standard methods, as described by Sambrook et al., Guide to Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (1989). The gene can be obtained either from monocot or dicot plant cells. When the gene encoding a plant acetyl CoA carboxylase is obtained from a dicot plant, the enzyme encoded by the gene exhibits tolerance to cyclohexanedione or aryloxyphenoxypropanoic acid herbicides. The gene can also be obtained from herbicide-tolerant maize cell lines, prepared as described in U.S. Pat. No. 5,162,602.

A gene encoding a plant acetyl CoA carboxylase can be identified by screening of a DNA or cDNA library generated from plant cells. Screening for DNA fragments that encode all or a portion of the gene encoding a plant acetyl CoA carboxylase can be accomplished by complementation of an auxotrophic mutant of acetyl CoA carboxylase in E. coli (fabE) (Bachman, Microbiological Reviews, 47:180 (1983)) or veast (acc1) (Michionada, Eur. J. Biochem., 111:79 (1980)) or by screening of plaques for binding to antibodies that specifically recognize a plant acetyl CoA carboxylase. DNA fragments that can restore ACCase activity in E. coli or yeast and/or plaques carrying DNA fragments that are immunoreactive with antibodies to a plant ACCase can be subcloned into a vector and sequenced and/or used as probes to identify other cDNA or genomic sequences encoding all or a portion of a plant acetyl CoA carboxylase gene.

Specific examples of cDNA sequences encoding a portion of a plant acetyl CoA carboxylase gene include DNA fragments that include a DNA sequence that substantially corresponds to the coding sequence for the transcarboxylase active site of a plant acetyl CoA carboxylase, DNA fragments that include a DNA sequence that substantially corresponds to a coding sequence for the biotin binding site of a plant acetyl CoA carboxylase, a DNA fragment encoding the 5′ transcriptional start sequence of a plant acetyl CoA carboxylase gene, and a DNA fragment encoding the 3′ transcriptional stop sequence for the acetyl CoA carboxylase gene. Substantially corresponding DNA sequences share about 90% to about 100% DNA sequence homology. Especially preferred cDNA probes can be obtained from lambda clone #18-5 which include DNA sequences corresponding to the transcarboxylase active site domain and the biotin binding site domain. Lambda clone #18-5 includes EcoRI subclones of 3.9 kb, 1.2 kb, or 0.23 kb. Lambda subclone #18-5I is an 3.9 kb EcoRI subclone. The lambda subclone #18-5I has been deposited with the American Type Culture Collection, Rockville, Md., and given Accession No. 69236.

In a preferred version, a plant acetyl CoA carboxylase gene is identified and isolated from an herbicide tolerant maize cell line prepared as described in Example II. A cDNA library can be prepared by oligo dT priming. Plaques containing DNA fragments can be screened with antibodies specific for maize acetyl CoA carboxylase. DNA fragments encoding a portion of an acetyl CoA carboxylase gene can be subcloned and sequenced and used as probes to identify a genomic acetyl CoA carboxylase gene. DNA fragments encoding a portion of a maize acetyl CoA carboxylase can be verified by, determining sequence homology with other known acetyl CoA carboxylases, such as chicken or yeast acetyl CoA carboxylase, or by hybridization to acetyl CoA carboxylase specific messenger RNA. Once DNA fragments encoding portions of the 5′, middle and 3′ ends as well as the transcarboxylase active site or biotin binding site of a plant acetyl CoA carboxylase are obtained, they can be used to identify and clone a complete genomic copy of a maize acetyl CoA carboxylase gene.

To isolate a complete copy of a maize acetyl CoA carboxylase gene, a maize genomic library can then be probed with cDNA probes prepared as described above. Portions of the genomic copy or copies of a plant acetyl CoA carboxylase gene can be sequenced and the 5′ end of the gene are identified by standard methods including either DNA sequence homology to other acetyl CoA carboxylase genes or by RNAase protection analysis, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989). Once portions of the 5′ end of the gene are identified, complete copies of a plant acetyl CoA carboxylase gene can be obtained by standard methods, including by cloning or by polymerase chain reaction (PCR) synthesis using oligonucleotide primers complementary to the DNA sequence at the 5′ end of the gene. The presence of an isolated full-length copy of a plant acetyl CoA carboxylase gene can be verified by hybridization, partial sequence analysis, or by expression of a plant acetyl CoA carboxylase enzyme. The maize acetyl CoA carboxylase gene cloned and expressed from a maize herbicide tolerant cell line can be assessed for tolerance to cyclohexanedione or aryloxyphenoxypropanoic acid herbicides by standard methods, as described in Example I.

An expression cassette of the invention can also contain an antisense DNA sequence. A antisense DNA sequence is a sequence that is substantially complementary to all or a portion of a coding sequence of a plant acetyl CoA carboxylase gene. A substantially complementary sequence has about 90% to about 100% DNA sequence homology with that of the coding sequence of all or a portion of a plant acetyl CoA carboxylase. The antisense DNA sequence when expressed can act to inhibit the synthesis and expression of a native plant acetyl CoA carboxylase. Antisense sequences are preferably about 200 to 1000 nucleotides long in order to provide sufficient inhibition of synthesis and/or expression of a native acetyl CoA carboxylase. The inhibition of acetyl CoA carboxylase synthesis and gene expression by antisense DNA sequences can be confirmed in a transformed plant cell by standard methods for measuring the presence and/or activity of the enzyme such as described in Examples I and V.

An expression cassette of the invention can also include a functional mutant of a plant acetyl CoA carboxylase gene. Mutants of a plant acetyl CoA carboxylase gene are substantially homologous to a plant acetyl CoA carboxylase gene and are functional if the acetyl CoA carboxylase expressed retains significant enzyme activity. A mutant substantially homologous to a plant acetyl CoA carboxylase can share about 90% to 99.99% DNA sequence with that gene. For example, a mutant acetyl CoA carboxylase gene can code for a herbicide tolerant acetyl CoA carboxylase, or for an acetyl CoA carboxylase with altered substrate specificity so that the total amount of oil content in the plants or seeds is increased, or for an enzyme with an altered substrate specificity so that synthesis of secondary metabolites such as flavonoids or anthocyanins is decreased. A preferred mutant is a gene coding for an acetyl CoA carboxylase that is substantially tolerant to cyclohexanedione or aryloxyphenoxypropanoic acid herbicide.

Altered or functional mutants of a gene coding for a plant acetyl CoA carboxylase can be obtained by several methods. The alteration or mutation of the ACCase gene can be accomplished by a variety of means including, but not limited to, the following methods.

1. spontaneous variation and direct mutant selection in cultures;

2. direct or indirect mutagenesis procedures on tissue culture of all cell types, seeds or plants; and

3. mutation of the cloned acetyl CoA carboxylase gene by methods such as site specific mutagenesis (Sambrook et al., cited supra), transposon mediated mutagenesis (Berg et al., Biotechnology, 1:417 (1983)) and deletion mutagenesis (Mitra et al., Molec. Gen. Genetic., 215:294 (1989)).

Mutants can be identified by a change in a functional activity of the enzyme. encoded by the gene or by detecting a change in the DNA sequence using restriction enzyme mapping or partial sequence analysis.

In a preferred version, a functional mutant gene encoding for a plant acetyl CoA carboxylase tolerant to cyclohexanedione and/or aryloxyphenoxypropanoic acid herbicides is isolated from a maize herbicide tolerant cell line. The maize herbicide tolerant cell line was obtained as described in U.S. Pat. No. 5,162,602, issued Nov. 10, 1992, the disclosure of which is incorporated in Examples I-III. Briefly, partially differentiated cell cultures are grown and subcultured with continuous exposures to low herbicide levels. Herbicide concentrations are then gradually increased over several subculture intervals. Maize cells or tissues growing in the presence of normally toxic herbicide levels are repeatedly subcultured in the presence of the herbicide and characterized. Stability of the herbicide tolerance trait of the cultured cells may be evaluated by growing the selected cell lines in the absence of herbicides for various periods of time and then analyzing growth after exposing the tissue to herbicide.

Maize cell lines which are tolerant by virtue of having an altered acetyl CoA carboxylase enzyme can be selected by identifying cell lines having enzyme activity in the presence of normally toxic levels of sethoxydim or haloxyfop. The tolerant maize cells can be further evaluated for whether acetyl CoA carboxylase is altered to a less sensitive form or increased in its level of expression.

Maize cell lines with a acetyl CoA carboxylase less sensitive to herbicide inhibition can be used to isolate a functional mutant gene of a plant acetyl CoA carboxylase. A DNA library from a maize cell line tolerant to herbicides can be generated and DNA fragments encoding all or a portion of an acetyl CoA carboxylase gene can be identified by hybridization to a cDNA probe encoding a portion of the maize ACCase gene. A complete copy of the altered gene can be obtained either by cloning and ligation or by PCR synthesis using appropriate primers. The isolation of the altered gene coding for acetyl CoA carboxylase can be confirmed in transformed plant cells by determining whether the acetyl CoA carboxylase being expressed retains enzyme activity when exposed to normally toxic levels of herbicides.

2. Promoters

Once a plant acetyl CoA carboxylase gene or functional mutant thereof or an antisense DNA sequence is obtained and amplified, it is combined with a promoter functional in a plant cell to form an expression cassette.

Most genes have regions of DNA sequence that are known as promoters and which regulate gene expression. Promoter regions are typically found in the flanking DNA sequence upstream from the coding sequence in both procaryotic and eukaryotic cells. A promoter sequence provides for regulation of transcription of the downstream gene sequence and typically includes from about 50 to about 2,000 nucleotide base pairs. Promoter sequences also contain regulatory sequences such as enhancer sequences that can influence the level of gene expression. Some isolated promoter sequences can provide for gene expression of heterologous genes, that is a gene different from the native or homologous gene. Promoter sequences are also known to be strong or weak or inducible. A strong promoter provides for a high level of gene expression, whereas a weak promoter provides for a very low level of gene expression. An inducible promoter is a promoter that provides for turning on and off of gene expression in response to an exogenously added agent or to an environmental or developmental stimulus. Promoters can also provide for tissue specific or developmental regulation. An isolated promoter sequence that is a strong promoter for heterologous genes is advantageous because it provides for a sufficient level of gene expression to allow for easy detection and selection of transformed cells and provides for a high level of gene expression when desired.

The promoter in an expression cassette of the invention can provide for overexpression of acetyl CoA of a plant acetyl CoA carboxylase gene or functional mutant thereof. Overexpression of the gene is that amount of gene expression that results in an increase in tolerance of the plant cells to an herbicide or that results in an increase in the total oil content of the cells. Overexpression of an acetyl CoA carboxylase gene is preferably about a 2- to 20-fold increase in expression of an acetyl CoA carboxylase over the expression level of the native acetyl CoA carboxylase. The promoter can also be inducible so that gene expression can be turned on or off by an exogenously added agent. For example a bacterial promoter such as the P_(tac) promoter can be induced to varying levels of gene expression depending on the level of isothiopropylgalactoside added to the transformed bacterial cells. It may also be preferable to combine the gene with a promoter that provides tissue specific expression or developmentally regulated gene expression in plants.

Specific promoters functional in plant cells include the 35S cauliflower mosaic virus promoter, nopaline synthase (NOS) promoter and the like. Currently, a preferred promoter for expression in monocots is the 35S cauliflower mosaic virus promoter.

An acetyl CoA carboxylase gene can be combined with the promoter by standard methods as described in Sambrook cited supra. Briefly, a plasmid containing a promoter such as the 35S cauliflower mosaic virus promoter can be constructed as described in Jefferson, Plant Molecular Biology Reporter, 5,387 (1987) or obtained from Clontech Lab in Palo Alto, Calif. (e.g. pBI121 or pBI221). Typically these plasmids are constructed to provide for multiple cloning sites having specificity for different restriction enzymes downstream from the promoter. A gene for plant acetyl CoA carboxylase can be subcloned downstream from the promoter using restriction enzymes to ensure that the gene is inserted in proper orientation with respect to the promoter so that the gene can be expressed, In a preferred version, a maize acetyl CoA carboxylase is operably linked to a 35 S CaMV promoter in a plasmid such as pBI121 or pBI221. Once a plant acetyl CoA carboxylase gene is operably linked to a promoter and the plasmid, the expression cassette so formed can be subcloned into other plasmids or vectors.

3. Optional Sequences in the Expression Cassette

The expression cassette can also optionally contain other DNA sequences. The expression cassette can further be comprised of a chloroplast transit peptide sequence operably linked between a promoter and a plant acetyl CoA carboxylase gene. If the expression cassette is to be introduced into a plant cell, the expression cassette can also contain plant transcriptional termination and polyadenylation signals and translational signals linked to the 3′ terminus of a plant acetyl CoA carboxylase gene. The expression cassette can also optionally be further comprised of a plasmid.

Because one site of action for biosynthetic pathways involving plant acetyl CoA carboxylase is the chloroplast, an expression cassette of the invention can be combined with an exogenous DNA sequence coding for a chloroplast transit peptide, if necessary. An exogenous chloroplast transit peptide is one which is not encoded within the plant acetyl CoA carboxylase gene. A chloroplast transit peptide is typically 40 to 70 amino acids in length and functions post-translationally to direct the protein to the chloroplast. The transit peptide is cleaved either during or just after import into the chloroplast to yield the mature protein. The complete copy of a gene encoding a plant acetyl CoA carboxylase may contain a chloroplast transit peptide sequence. In that case, it may not be necessary to combine an exogenously obtained chloroplast transit peptide sequence into the expression cassette.

Exogenous chloroplast transit peptide encoding sequences can be obtained from a variety of plant nuclear genes, so long as the products of the genes are expressed as preproteins comprising an amino terminal transit peptide and transported into chloroplast. Examples of plant gene products known to include such transit peptide sequences are the small subunit of ribulose biphosphate carboxylase, ferredoxin chlorophyll a/b binding, protein, chloroplast ribosomal proteins encoded by nuclear genes, certain heatshock proteins, amino acid biosynthetic enzymes such as acetohydroxy acid synthase, 3-enolpyruvylphosphoshikimate synthase, dihydrodipicolinate synthase, and the like. Alternatively, the DNA fragment coding for the transit peptide may be chemically synthesized either wholly or in part from the known sequences of transit peptides such as those listed above.

Regardless of the source of the DNA fragment coding for the transit peptide, it should include a translation initiation codon and an amino acid sequence that is recognized by and will function properly in chloroplasts of the host plant. Attention should also be given to the amino acid sequence at the junction between the transit peptide and the plant acetyl CoA carboxylase enzyme where it is cleaved to yield the mature enzyme. Certain conserved amino acid sequences have been identified and may serve as a guideline. Precise fusion of the transit peptide coding sequence with the acetyl CoA carboxylase coding sequence may require manipulation of one or both DNA sequences to introduce, for example, a convenient restriction site. This may be accomplished by methods including site directed mutagenesis, insertion of chemically synthesized oligonucleotide linkers and the like.

Once obtained, the chloroplast transit peptide sequence can be appropriately linked to the promoter and a plant acetyl CoA carboxylase gene in an expression cassette using standard methods. Briefly, a plasmid containing a promoter functional in plant cells and having multiple cloning sites downstream can be constructed as described in Jefferson cited supra. The chloroplast transit peptide sequence can be inserted downstream from the promoter using restriction enzymes. A plant acetyl CoA carboxylase gene can then be inserted immediately downstream from and in frame with the 3′ terminus of the chloroplast transit peptide sequence so that the chloroplast transit peptide is linked to the amino terminus of the plant acetyl CoA carboxylase. Once formed, the expression cassette can be subcloned into other plasmids or vectors.

When the expression cassette is to be introduced into a plant cell, the expression cassette can also optionally include 3′ nontranslated plant regulatory DNA sequences. The 3′ nontranslated regulatory DNA sequence preferably includes from about 300 to 1,000 nucleotide base pairs and contains plant transcriptional and translational termination sequence. Specific examples of 3′ nontranslated regulatory DNA sequences functional in plant cells include about 500 base pairs of the 3′ flanking DNA sequence of the pea ribulose biphosphate carboxylase small subunit E9 gene, the 3′ flanking DNA sequence of the octopine synthase gene, and the 3′ flanking(DNA sequence of the nopaline synthase gene. These 3′ nontranslated regulatory sequences can be obtained as described in An, Methods in Enzymology, 153:292 (1987) or are already present in plasmids available from commercial sources such as Clontech, Palo Alto, Calif. The 3′ nontranslated regulatory sequences can be operably linked to the 3′ terminus of a plant acetyl CoA carboxylase gene by standard methods.

An expression cassette of the invention can also be further comprised of a plasmid. Plasmid vectors included additional DNA sequences that provide for easy selection, amplification and transformation of the expression cassette in procaryotic and eukaryotic cells. The additional DNA sequences include origins of replication to provide for autonomous replication of the vector, selectable marker genes, preferably encoding antibiotic resistance, unique multiple cloning sites providing for multiple sites to insert DNA sequences or genes encoded in the expression cassette, and sequences that enhance transformation of prokaryotic and eukaryotic cells. The preferred vectors of the invention are plasmid vectors. The especially preferred vector is the pBI121 or pBI221 vector formed as described by Jefferson cited supra.

Another vector that is useful for expression in both plant and procaryotic cells is the binary Ti vector PGA582. This binary Ti vector has been previously characterized by An, cited supra., and is available from Dr. An. This binary Ti vector can be replicated in procaryotic bacteria such as E. coli and Agrobacterium. The Agrobacterium plasmid vectors can be used to transfer the expression cassette to plant cells. The binary Ti vectors preferably include the nopaline T DNA right and left borders to provide for efficient plant cell transformation, a selectable marker gene, unique multiple cloning sites in the T border regions the colE1 replication of origin and a wide host range replicon. The binary Ti vectors carrying an expression cassette of the invention can be used to transform both prokaryotic and eukaryotic cells, but is preferably used to transform plant cells.

B. Method for Screening for Expression and/or Overexpression of a Plant Acetyl CoA Carboxylase Gene

A method for screening for expression or overexpression of a plant acetyl CoA carboxylase gene is also provided by the invention. Once formed, an expression cassette comprising an acetyl CoA carboxylase gene can be subcloned into a known expression vector. The screening method in the invention includes the steps of introducing an expression vector into a host cell and detecting and/or quantitating expression of a plant acetyl CoA carboxylase gene. This method of screening is useful to identify expression cassettes providing for an overexpression of a plant acetyl CoA carboxylase gene, antisense molecules that effectively inhibit acetyl CoA carboxylase synthesis, and expression of an acetyl CoA carboxylase in the chloroplast of a transformed plant cell.

Suitable known expression vectors include plasmids that autonomously replicate in prokaryotic and eukaryotic cells. Specific examples include plasmids such as the pBI121 or pBI221 plasmid constructed as described by Jefferson cited supra, a binary Ti vector such as PG582 as described by An cited supra, PUC119, or PBR322. The preferred expression system is a pBI121 or pBI221 plasmid.

An expression cassette of the invention can be subcloned into an expression vector by standard methods. The expression vector can then be introduced into prokaryotic or eukaryotic cells by standard methods including protoplast transformation, Agrobacterium mediated transformation, electroporation, microprojectiles and liposomes. The expression vector can be introduced into plant cells such as tobacco, Brassica, Black Mexican sweet corn, and Arabidopsis cells. The vector can also be introduced into procaryotic cells such as E. coli or Agrobacterium. Transformed cells can be selected typically using a selection marker encoded on the expression vector.

Transient expression of a plant acetyl CoA carboxylase gene can be detected and quantitated in the transformed cells. Gene expression can be quantitated by a quantitative Western blot using antibodies specific for the cloned acetyl CoA carboxylase or by detecting an increase in specific activity of the enzyme. The tissue and subcellular location of the cloned acetyl CoA carboxylase can be determined by immunochemical staining methods using antibodies specific for the cloned acetyl CoA carboxylase. Sensitivity of the cloned acetyl CoA carboxylase to herbicides can also be assessed. Expression cassettes providing for overexpression of a plant acetyl CoA carboxylase or acetyl CoA carboxylase tolerant to herbicides can then be used to transform monocot and/or dicot plant tissue cells and to regenerate transformed plants and seeds.

C. Method of Imparting Cyclohexanedione or Aryloxyphenoxypropanoic Acid Herbicide Tolerance to a Plant

The invention provides a method of conferring cyclohexanedione or aryloxyphenoxypropanoic acid herbicide tolerance to a plant. The method includes the steps of introducing an expression cassette comprising a gene coding for a plant acetyl CoA carboxylase or a functional mutant thereof operably linked to a promoter into the cells of plant tissue and expressing the gene in an amount effective to render the cells of the plant tissue substantially tolerant to herbicides. An effective amount of gene expression to render the cells of the plant tissue substantially tolerant to the herbicide depends on whether the gene codes for an unaltered acetyl CoA carboxylase gene or a mutant or altered form of the gene that is less sensitive to the herbicides. Expression of an unaltered plant acetyl CoA carboxylase gene in an effective amount is that amount that provides for a 2- to 50-fold increase in herbicide tolerance and preferably increases the amount of acetyl CoA carboxylase from at least about 2-to 20-fold over that amount of the native enzyme. An altered form of the enzyme can be expressed at levels comparable to that of the native enzyme or less if the altered form of the enzyme has higher specific activity. Acetyl CoA carboxylase substantially tolerant to herbicides is an enzyme that is tolerant of levels of herbicide which normally inhibit a native acetyl CoA carboxylase and preferably can function in concentrations of herbicide of about 2- to 20-fold greater than are toxic to the native enzyme.

Herbicide tolerance can be achieved by at least two methods including: 1) by increasing the level of gene expression of a native or unaltered acetyl CoA carboxylase gene; or 2) by introducing an altered gene coding for an acetyl CoA carboxylase that is less sensitive to herbicide inhibition. The level of gene expression can be increased by either combining a plant acetyl CoA carboxylase gene with a promoter that provides for a high level of gene expression such as the 35S CaMV promoter or by introducing the gene into the cells so that multiple copies of the gene are integrated into the genome of the transformed plant cell. Formation of an expression cassette comprised of a plant acetyl CoA carboxylase gene operably linked to a promoter that can be expressed in an effective amount to confer herbicide tolerance has been described previously.

Most monocots, but not dicots, are sensitive to cyclohexanedione and/or aryloxyphenoxypropanoic acid herbicides. The preferred plant cells for introducing an expression cassette of the invention to achieve herbicide tolerance for the plant cells then are monocot plants. Monocot plants include corn, wheat, barley, sorghum, rice, and others. An expression cassette of the invention can be introduced by methods of transformation, especially effective for monocots including biolistic transformation of Type II embryogenic suspension cells as described by W. J. Gordon-Kamm et al., Plant Cell, 2, 603-618 (1990), M. E. Fromm et al., Bio/Technology, 8, 833-839 (1990) and D. A. Walters et al., Plant Molecular Biology, 18, 189-200 (1992) or by electroporation of type I embryogenic calluses described by D'Hafluin et al., The Plant Cell, 4, 1495 (1992). Transformed cells can be selected for the presence of a selectable marker gene. Transient expression of a plant acetyl CoA carboxylase gene can be detected in the transgenic embryogenic calli using antibodies specific for the cloned plant acetyl CoA carboxylase. Transformed embryogenic calli can be used to generate transgenic plants that exhibit stable inheritance of either the altered acetyl CoA carboxylase gene or overexpression of the acetyl CoA carboxylase gene. Maize cell lines exhibiting satisfactory levels of tolerance to herbicide are put through a plant regeneration protocol to obtain mature maize plants and seeds expressing the tolerance traits such as described in D'Hafluin. cited supra., or An, cited supra. The plant regeneration protocol allows the development of somatic embryos and the subsequent growth of roots and shoots. To determine that the herbicide-tolerance trait is expressed in differentiated organs of the plant, and not solely in undifferentiated cell culture, regenerated plants are exposed to herbicide levels which will normally inhibit shoot and root formation and growth.

Mature maize plants are then obtained from maize cell lines that are known to express the trait. If possible, the regenerated plants are self-pollinated. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important inbred lines. Conversely, pollen from plants of these inbred lines is used to pollinate regenerated plants. The genetics of the trait are then characterized by evaluating the segregation of the trait in the first and later generation progeny. Stable inheritance of overexpression of a plant acetyl CoA carboxylase or a functional mutant of a plant acetyl CoA carboxylase conferring herbicide tolerance to the plant is achieved if the plants maintain herbicide tolerance for at least about three to six generations.

Seed from transformed monocot plants regenerated from transformed tissue cultures is grown in the field and self-pollinated to generate true breeding plants. Progenies from these plants become true breeding lines which are evaluated for herbicide tolerance in the field under a range of environmental conditions. Herbicide tolerance must be sufficient to protect the monocot plants at the maximum labeled delivery rate under field conditions which cause herbicides to be most active. Appropriate herbicide concentrations and methods of application are those which are known and have been developed for the cyclohexanedione and/or aryloxyphenoxypropanoic acid herbicides in question.

In a preferred version, an expression cassette comprised of a maize acetyl CoA carboxylase gene isolated from a maize cell line tolerant to sethoxydim and haloxyfop and linked to the 35S CaMV promoter is introduced into an herbicide sensitive monocot tissue using biolistic transformation. Transformed calli are selected and used to generate transgenic plants. Transformed calli and transgenic plants can be evaluated for tolerance to sethoxydim and haloxyfop and for stable inheritance of the tolerance trait.

D. Method for Altering the Oil Content in a Plant

The invention also provides a method of altering the oil content in a plant. The method include the steps of introducing an expression cassette comprising a gene coding for plant acetyl CoA carboxylase or functional mutant thereof operably linked to a promoter functional in a plant cell into the cells of plant tissue and expressing the gene in an amount effective to alter the oil content of the plant cell. An alteration in the oil content of a plant cell can include a change in the total oil content over that normally present in that type of plant cell, or a change in the type of oil from that normally present in the plant cell. Expression of the gene in an amount effective to alter the oil content of the gene depends on whether the gene codes for an unaltered acetyl CoA carboxylase or a mutant or altered form of the gene. Expression of an unaltered plant acetyl CoA carboxylase gene in an effective amount is that amount that may provide a change in the oil content of the cell from about 1.2- to 20-fold over that normally present in that plant cell, and preferably increases the amount of acetyl CoA carboxylase about 2- to 20-fold over that amount of the enzyme normally present in that plant cell. An altered form of the enzyme can be expressed at levels comparable to that of the native enzyme or less if the altered form of the enzyme has higher specific activity.

An alteration in oil content of the plant cells according to the method of the invention can be achieved in at least two ways including:

(1) an increase or decrease in expression of an unaltered plant acetyl CoA carboxylase gene; or

(2) by introducing an altered or functional mutant plant acetyl CoA carboxylase gene coding for an enzyme that exhibits a change in specific activity.

The level of gene expression of an unaltered plant acetyl CoA carboxylase gene can be increased by either combining an unaltered plant acetyl CoA carboxylase gene with a promoter that provides for a high level of gene expression, such as the 35S cauliflower mosaic virus or by introducing the expression cassette and/or selecting for plant cells having multiple copies of a plant acetyl CoA carboxylase gene integrated into the genome. A decrease in expression of an unaltered acetyl CoA carboxylase can be achieved by transformation with an ACCase antisense gene containing an expression cassette. When an altered or functional mutant plant acetyl CoA carboxylase gene codes for an enzyme that has an increase in specific activity, it may lead to an increase in total oil content of a plant cell even if the level of gene expression is comparable to that of the native enzyme. When an altered or functional mutant acetyl CoA carboxylase gene codes for an enzyme having a decrease in specific activity, it may lead to a decrease in the total oil content of the plant cell compared to that normally present.

An expression cassette as described above can be introduced into either monocots or dicots. Preferably, the expression cassette is introduced into dicot plants such as soybean, canola, and sunflower. An expression cassette can be introduced by standard methods including protoplast transformation, Agrobacterium-mediated transformation, microprojectiles, electroporation, and the like. Transformed cells or tissues can be selected for the presence of a selectable marker gene.

Transient expression of a plant acetyl CoA carboxylase gene can be detected in transformed cells or tissues by immunoreactivity with antibodies specific for the cloned acetyl CoA carboxylase. Overexpression of a plant acetyl CoA carboxylase can be detected by quantitative Western blots. A change in specific activity of the enzyme can be detected by measuring enzyme activity in the transformed cells. A change in total oil content can also be examined by standard methods, as described in Clark & Snyder, JACS, 66:1316 (1989).

Transgenic plants and seeds can be generated from transformed cells and tissues showing a change in oil content or in the amount or specific activity of a plant acetyl CoA carboxylase using standard methods. It is especially preferred that the oil content of the leaves, seeds, or fruits is increased.

In a preferred version a maize acetyl CoA carboxylase gene is combined with a 35S cauliflower mosaic virus promoter in a vector such as pBI121 or pBI221 and introduced into soybean cells using the microprojectile method. Transformed soybean cells showing an increase in expression of acetyl CoA carboxylase of at least about 2-fold or at least a 1.2-fold increase in oil content are selected. Transformed soybean cells exhibiting overexpression of acetyl CoA carboxylase or showing an increase in total oil content are used to generate transgenic plants and seeds.

E. Method of Producing Plant Acetyl CoA Carboxylase

The invention also provides a method of producing plant acetyl CoA carboxylase in a host cell. The method includes the steps of introducing an expression cassette comprised of a gene encoding a plant acetyl CoA carboxylase or functional mutant thereof into a host cell and expressing the gene in an amount sufficient to allow for crystallization of the plant acetyl CoA carboxylase. An amount sufficient to allow for crystallization of a plant acetyl CoA carboxylase is about 20- to 100-fold increase over the amount of plant acetyl CoA carboxylase that can normally be purified from plant cells, preferably about 2 to 10 mg protein. Crystallized plant acetyl CoA carboxylase can be used to identify other herbicides that can bind to and inhibit acetyl CoA carboxylase function. In addition, the availability of large amounts of purified enzyme provides for screening of the efficacy of such herbicides.

An expression cassette can include a promoter that is functional in either a eukaryotic or prokaryotic cell. The expression cassette can be introduced into a prokaryotic cell such as E. coli, or a eukaryotic cell such as a plant or yeast. The preferred cell is a prokaryotic cell used routinely in producing recombinant proteins such as E. coli. The expression cassette can be introduced and transformed cells selected by standard methods.

The plant acetyl CoA carboxylase gene can be expressed in an prokaryotic cell until sufficient amount of the enzyme is produced so that it can be crystallized. Plant acetyl CoA carboxylase can be isolated from bacterial cells using standard methods, including those described in Example V. The purified acetyl CoA carboxylase can then be crystallized and characterized by standard methods.

EXAMPLE I Identification of Herbicide Mechanism and Site of Action

The objective of this Example was to identify the mechanism whereby sethoxydim and/or haloxyfop inhibit fatty acid synthesis in maize. The results, reported in J. D. Burton et al., Biochem. Biophys. Res. Comm., 148, 1039 (Nov. 13, 1987), show that both sethoxydim and haloxyfop inhibit acetyl-coenzyme A carboxylase (ACCase) (EC 6.4.1.2) in maize chloroplasts.

A. Chemicals

Buffers and cofactors were purchased from Sigma Chemical Company (St. Louis, Missouri); [2-¹⁴C]acetate was purchased from Research Products International; [2-¹⁴C]pyruvate and [¹⁴C]NaHCO₃ were purchased from New England Nuclear; and [2-¹⁴C]malonyl coenzyme A was purchased from Amersham. Sethoxydim was a gift from BASF (Parsippany, N.J.), and haloxyfop was provided by Dow Chemical USA (Midland, Mich.).

B. Plant Growth Conditions

Corn (Z. mays L., ‘B37×Oh43’) seeds were germinated in darkness for 96 hours in vermiculite in an incubation chamber maintained at 30° C., 80% RH. Seedlings were then transferred to a growth chamber with a 16 hour light (25° C.) and an 8 hour dark (20° C.) cycle, 90% relative humidity (RE). After greening 48 hours, seedlings were returned to the dark incubation chamber for 12 hours to deplete chloroplast starch reserves. Seedlings were harvested 6 days after planting. Pea (P. sativum L., ‘PI 9901-C’) seedlings were grown in vermiculite in a growth chamber with a 16 hour light (21° C.) and 8 hour dark (16° C.) cycle, 80% RH. Peas were harvested 10 to 13 days after planting. Black Mexican Sweet (BMS) corn suspension cultures ware maintained in a supplemented Murashige-Skoog (MS) medium (C. E. Green. Hort. Sci., 12. 7-10 (1977)), and subcultured weekly by 20-fold dilution of the suspension culture into fresh medium.

C. Chloroplast Isolation

Chloroplasts from corn and pea seedlings there isolated at 4° C. (K. Cline et al., J. Biol. Chem., 260, 3691-3696 (1985)). Seedlings (50 g of shoots) were homogenized in 200 ml buffer A (50 mM HEPES-NaOH pH 7.5, 330 mM sorbitol, 0.1% w/v BSA, 1 mM MgCl₂, 1 mM MnCl₂, 2 mM EDTA, 5 mM isoascorbate, 1.3 mM glutathione) in an omnimixer (five. 3-second bursts at full speed). The homogenate was filtered through six layers of cheesecloth and two layers of miracloth, and then centrifuged at 3000 g for 3 minutes with hand-braking. The pellet was gently resuspended in buffer A and layered onto a preformed linear Percoll gradient (50 mM HEPES-NaOH pH 7.5, 330 mM sorbitol, 1.9 mM isoascorbate, 1.08 mM glutathione, 0.1% w/v BSA, 50% Percoll) which was centrifuged at 3000 g for 20 minutes in a Sorvall HB-4 rotor. The lower band in the gradient, containing intact chloroplasts, was washed twice by gently resuspending it in 20 ml of buffer B (50 mM HEPES-NaOH, pH 7.5, and 330 mM sorbitol) followed by repelleting (3000 g, 5 minutes). The final pellet, consisting of intact chloroplasts, was resuspended in 2 to 3 ml of buffer B and stored on ice in the dark until use.

D. Fatty Acid Synthesis

[¹⁴C]acetate and [¹⁴C]pyruvate were used as precursors to measure fatty acid biosynthesis in isolated chloroplasts (B. Liedvogel et al., Planta, 169, 481-489 (1986)). [¹⁴C]acetate incorporation was assayed in a 0.5 ml-volume containing: 50 mM HEPES-NaOH (pH 7.5), 330 mM sorbitol, 5 mM KH₂PO₄, 10 mM NaHCO₃, 1 mM MgCl₂, 1 mM ATP, 0.1 mM CoA, 0.15 mM [¹⁴C]acetate (3.33 mCi/mmol), and chloroplasts (20 to 50 μg chlorophyll). [¹⁴C]pyruvate incorporation into fatty acids was assayed in the same medium except that it included 2 mM TPP, 1 mM NAD⁻. 0.15 mM [¹⁴C]-pyruvate (1.33 mCi/mmol), but no acetate. Assay suspensions were illuminated with 1400 μE/m²·second PAR at 25° C. Assays were initiated by the addition of the labeled substrate and stopped by the addition of 0.5 ml ol 40% KOH. To determine the incorporation of radiolabel into a non-polar (fatty acid) fraction, each treatment was saponified at 90° C. for 30 minutes in capped vials (P. B. Hoj et al., Carlsberg Res. Commun., 47, 119-141 (1982)). The vials were acidified with 0.5 ml 40% H₂SO₄, and carrier fatty acids (20 μg each of C 14:0, C 16:0, and C 18:0) were added. The assay mixture was extracted twice with 4 ml hexane. The extracts were combined, dried under N₂, and redissolved in 0.3 ml hexane. Aliquots (50 μl) were counted for radioactivity by liquid scintillation spectrometry.

Incorporation of [¹⁴C]malonyl-Coenzyme A into fatty acids (P. B. Hoj et al., supra; and J. B. Ohlrogge et al., Proc. Natl. Acad. Sci. USA, 76, 1194-1198 (1979)) was assayed using cell-free preparations from BMS tissue culture. Cells harvested during logarithmic growth phase were frozen in liquid nitrogen, ground with a mortar and pestle, and thawed in a medium containing: 0.1 M HEPES-KOH, pH 7.5; 0.3 M glycerol, and 5 mM DTT (buffer:tissue, 2:1, v/w). The homogenate was centrifuged at 12,000 g for 20 minutes. The supernatant was filtered through miracloth and centrifuged (125,000 g) for 60 minutes and then filtered through miracloth and assayed. Assays were conducted at 25° C. in a 0.4 ml volume containing: 1.0 mM ATP, 0.32 mM NADPH, 0.38 mM NADH, 25 μM CoA, 10 μM acetyl-CoA, 25 μg acyl-carrier protein, and 12 μM malonyl-CoA (11.54 μCi/μmol). Reactions were initiated by addition of [¹⁴C]malonyl CoA and stopped by addition of 0.4 ml 40% KOH. Label incorporation into fatty acids was determined as above. Chlorophyll (D. I. Arnon, Plant Physiol., 24, 1-15 (1949)) and protein (P. K. Smith et al., Anal. Biochem., 150, 76-85 (1985)) were determined as described therein.

E. Acetyl-Coenzyme A Carboxylase (ACCase) Activity

Maize chloroplasts, isolated as described above, were suspended in buffer C (0.1 M Tricine-KOH, pH 8.0; 0.3 M glycerol, and 1 mM DTT) and homogenized in a glass tissue homogenizer. The disrupted chloroplast fraction was centrifuged at 16,000 g for 15 minutes. The supernatant was desalted on a Sephadex G-25 column (1.5×5 cm equilibrated with 0.1 M Tricine-KOH, pH 8.0: and 0.3 M glycerol) and assayed directly. ACCase activity (B. J. Nikolau et al., Arch. Biochem. Biophys., 211, 605-612 (1981)) was assayed at 30° C. in a 0.2 ml volume which contained 1 mM ATP, 3 mM acetyl coenzyme A, 2.5 mM MgCl₂, 50 mM KCl, 0.5 mM DTT, and 15 mM [¹⁴C]NaHCO₃ (0.17 mCi/mmol). Reactions were initiated by addition of acetyl coenzyme A and stopped by addition of 25 μl of 12 N HCl. Product formation was determined by the radioactivity found in an acid stable fraction by liquid scintillation spectrometry. Enzyme activity was linear for 15 minutes.

F. Results

To probe for the site of herbicidal activity of sethoxydim and haloxyfop, labeled acetate, pyruvate, and malonyl-CoA were used individually as precursors for fatty acid synthesis. Isolated chloroplasts from corn seedlings incorporated [¹⁴C]acetate and [¹⁴C]pyruvate into a non-polar fraction (fatty acids). Acetate incorporation was linear for 30 min after a 5 min lag period, and dependent upon the addition of free acetyl coenzyme A. Addition of either 10 μM sethoxydim or 1 μM haloxyfop inhibited [¹⁴C]acetate incorporation into fatty acids by 90% and 89%, respectively, as shown in Table I, below. Sethoxydim (10 μM) and haloxyfop (1 μM) also inhibited the incorporation of [¹⁴C]pyruvate into fatty acids by 98% and 99%, respectively.

TABLE I Inhibition of [¹⁴C]acetate and [¹⁴C]pyruvate Incorporation into Fatty Acids in Corn Seedling Chloroplasts by Sethoxydim (10 μM) and Haloxyfop (1 μM), 10 minute assay time Acetate Pyruvate Activity (nmol/mg chl · min) Control  4.4 ± 0.4¹ 10.8 ± 2.3   % Inhibition Sethoxydim  90 ± 2.5 98 ± 1.1 Haloxyfop  89 ± 3.1 99 ± 0.3 ¹Results are expressed as mean of two experiments ± standard error.

The effect of 10 μM sethoxydim and 1 μM haloxyfop on [¹⁴C]malonyl-CoA incorporation into fatty acids was determined using cell-free extracts from corn suspension cultures. Neither sethoxydim (10 μM) nor haloxyfop (1μM) inhibited fatty acid synthetase activity. Thus, both herbicides inhibited fatty acid synthesis in intact chloroplasts from corn seedlings with either acetate or pyruvate as a precursor, but did not inhibit incorporation of malonyl-CoA into fatty acids. This suggests that ACCase which catalyzes the formation of malonyl-CoA is the site of action of these herbicides.

EXAMPLE II Selection and Characterization of Herbicide-tolerant Cell Lines

A selection protocol to identify and isolate herbicide-tolerant maize cells was developed to minimize the adverse effects of high herbicide concentrations on somatic embryo development and plant regeneration capacity. The procedure involved exposing tissue to gradually increasing concentrations of herbicide beginning with a sethoxydim concentration representing {fraction (1/20)}th of lethal dose and doubling the herbicide concentration at approximately two-week intervals until the lethal dose (10 μM sethoxydim) was reached. In this way, the herbicide was allowed to take effect slowly with continuous selection pressure, thus permitting herbicide-tolerant cells to accumulate over time while not affecting the potential for plant regeneration.

A. Selection of a Sethoxydim-Tolerant Cell Line

Many selections were carried out utilizing the selection protocol described in the preceding paragraph. The selection of one such sethoxydim-tolerant cell line that was identified and characterized is described below in detail.

Approximately 100 grams of vigorously growing regenerable, friable, embryogenic maize callus tissue established from an F₁ immature embryo resulting from the cross A188×B73 were transferred to agar-solidified maintenance medium (Armstrong and Green, Planta. 164. 207 (1985)) in petri plates containing 0.5 μM sethoxydim (BASF) (Parsippany, N.J.). This callus line was designated 2167-9/2160-154. Forty plates were prepared and five clumps of callus tissue weighing about 0.5 grams each were placed on each plate. The 0.5 μM sethoxydim concentration was chosen from growth inhibition studies to provide less than 10-20% growth inhibition during the first two weeks of herbicide exposure. After 14 days, 0.25-0.5 gram pieces of tissue showing vigorous growth rate and retention of embryogenic morphology (i.e., presence of somatic embryos) were subcultured on fresh medium containing 1.0 μM sethoxydim. Eighty plates containing five pieces of tissue per plate were prepared. For each subsequent transfer, all callus tissue showing growth and somatic embryo forming ability was placed on fresh media containing a two-fold increased sethoxydim concentration. Therefore, callus was transferred at two-week intervals to petri plates containing 0.5, 1.0, 2.0, 5.0 and 10.0 μM sethoxydim. During the course of the selection process, the total number of lines decreased as the herbicide-mediated growth inhibition became more intense. Cell lines exhibiting growth on 10 μM sethoxydim were designated as herbicide-tolerant and given an identification number. Two sethoxydim-tolerant lines were recovered that exhibited uninhibited growth at 10 μM sethoxydim. These lines were designated 2167-9/2160-154 S-1 and 2167-9/2160-154 S-2.

B. Characterization of Herbicide-Tolerant Maize Cell Line 2167-9/2160-154 S-2

Tolerant cell line 2167-9/2160-154 S-2 (“S-2”) was characterized to evaluate: (1) the magnitude of sethoxydim tolerance; (2) cross-tolerance of haloxyfop: and (3) the biochemical basis for the tolerance. Callus tissue from S-2 that had been maintained on 10 μM sethoxydim was transferred to media containing up to 100 μM sethoxydim. One-half gram of S-2 tissue was plated on a 7 cm filter paper as a lawn overlaying 50 ml agar-solidified culture medium containing 0. 0.5. 1.0, 2.0, 5.0, 10.0, 50.0 and 100 μM sethoxydim, and cultured for two weeks. Control cell line 2167-9/2160-154 was plated similarly on medium containing the same levels of sethoxydim. The results of this study are summarized in FIG. 2. The control cell line growth after two weeks was inhibited 50% at 1 μM sethoxydim. Growth of S-2 was not inhibited at 100 μM sethoxydim, indicating that S-2 was at least 100-fold more tolerant than the control callus line.

Growth of S-2 was inhibited with 0.65 μM haloxyfop, whereas the control cell line was inhibited 50% with 0.02 μM, indicating approximately a 30-fold increase in tolerance.

C. Acetyl-Coenzyme A Carboxylase (ACCase) Activity of Maize Cell Line S-2

Assays were conducted to determine if ACCase extracted from cell line S-2 was altered with respect to herbicide activity. ACCase activity of control tissue was 50% inhibited either by 1.5 μM sethoxydim, or by 0.25 μM haloxyfop. ACCase activity of S-2 tissue was inhibited 50% either by 70 μM sethoxydim, or by 1.8 μM haloxyfop, indicating at least 40-fold and 7-fold decreases in herbicide sensitivity on concentration basis, respectively.

EXAMPLE III Plant Regeneration and Production of Herbicide-Tolerant Seed

A. Plant Regeneration Protocol

Sixteen ca. 150 mg clumps of S-2 callus were transferred per 25×100 mm petri plate containing agar-solidified N6 basal salts and 6% sucrose and incubated 7-14 days in low light (20 μE m⁻² s⁻¹). Several plates containing callus on plant regeneration medium were prepared. Callus was transferred to agar-solidified Murashige-Skoog (MS) medium without hormones and incubated in high intensity light (200 μE m-2 s⁻¹) for shoot elongation. Developing plants (I-3 cm long) were isolated from the callus surface and transferred to magenta boxes containing agar-solidified MS salts, 2% sucrose with no hormones for two weeks of further growth. When plants reached the 2-3 leaf stage, they were transplanted to peat pots containing potting soil, and were incubated in the growth room until growing stably. Surviving plants were transferred to soil in 4″ diameter plastic pots and grown in the greenhouse.

B. Expression of Herbicide Tolerance in Plants Regenerated from S-2 Callus Tissue

Groups of eight control (2167-9/2160-154 unselected) and eight S-2 plants were sprayed with either 0.0, 0.01. 0.05, 0.11, 0.22 or 0.44 kg/ha sethoxydim to determine whole plant sethoxydim-tolerance of greenhouse-grown plants. Control plants were killed by 0.05 kg/ha or more sethoxydim. Plants regenerated from the S-2 cell line survived the 0.44 kg/ha sethoxydim treatment, indicating that S-2 plants exhibit at least 20-fold more tolerance of sethoxydim than control. FIG. 3 shows the growth response of the regenerated plants seven days after treatment with 0.44 kg/ha sethoxydim. As shown in FIG. 4, shoot height of regenerated S-2 plants was only slightly reduced 14 days after treatment with 0.44 kg/ha sethoxydim.

C. Seed Production from S-2 Plants

Plants surviving sethoxydim treatments of up to 0.44 kg/ha were transplanted to the genetics plot on the University of Minnesota campus, St. Paul, Minnesota. Additional S-2 plants were transplanted to the field that had not been sprayed. Sixty-five 2167-9/2160-154 control plants and ninety-five S-2 plants were grown to maturity in the field. Plants were either self-pollinated or cross-pollinated to inbred maize lines A188, A619, A641. A661, A665, B37, B73, R806, and W153R. Control seed were produced by selfing 2167-9/2160-154 regenerated plants, or by crossing them with the inbreds listed above.

D. Expression of Herbicide Tolerance in Progeny of Regenerated Plants

Seeds obtained by the crossing procedure described above were viable and germinated normally. Seeds from thirty S-2 selfed plants and fifteen 2167-9/2160-154 control plants were planted in 25×50 cm trays of soil (28 seeds from each plant in one tray) and grown in the greenhouse. Seedlings at the 3-4 leaf stage were treated with 0.1, 0.44, and 1.1 kg/ha sethoxydim and evaluated for visual herbicide damage and shoot height. Based on visual rating of herbicide damage two weeks after treatment, selfed progeny of S-2 plants segregated approximately 1:2:1 for healthy, uninjured plants: to plants showing partial injury: to dead plants, respectively at 0.44 and 1.1 kg/ha sethoxydim treatments. All control progeny of 2167-9/2160-154 control plants were killed by 0.1 kg/ha and greater levels of sethoxydim. These results demonstrate dominant expression of sethoxydim tolerance indicating that sethoxydin tolerance in S-2 plants is a heritable trait. Similar tests were conducted on progeny of S-2 plants crossed to the other inbreds. In all cases, these test cross progeny treated with 0.44 kg/ha sethoxydim segregated 1:1 for growing shoots versus dead shoots whether S-2 plants were used as male or female parents. These results confirm that sethoxydim tolerance is controlled by, a single dominant nuclear gene. In all cases, control plants crossed to the other inbreds were killed and therefore sethoxydim-sensitive.

E. Method for Obtaining Uniform Herbicide-Tolerant Seed

Progeny of S-2 plants surviving sethoxydim treatments of 0.44 and 1.1 kg/ha and showing no herbicide injury were transferred to the greenhouse and grown to maturity. These plants may be selfed and their progeny evaluated for sethoxydim and haloxyfop tolerance to identify pure breeding herbicide-tolerant maize lines.

Progeny of S-2 plants crossed to inbred lines and exhibiting sethoxydim tolerance may be recurrently backcrossed to the same inbreds. Progeny of each cross may be screened for sethoxydim-tolerance, and tolerant plants grown to maturity and again crossed to the recurrent parent. After six or seven cycles of backcrossing, sethoxydim-tolerant plants may be selfed and progeny screened for tolerance to produce homozygous sethoxydim tolerant maize inbreds.

EXAMPLE IV Selection of Additional Herbicide-Tolerant Maize Cell Lines

One primarily sethoxydim-tolerant maize cell line, 2167-9/2160-154 S-1. and two haloxyfop-tolerant maize cell lines. 2167-9/2160-154 H-1 and 2167-9/2160-154 H-2. were selected and characterized as follows:

A. Selection of Maize Cell Line 2167-9/2160-154 S-1

Maize cell line 2167-9/2160-154 S-1 was selected from maize cell culture using the protocol described in detail above for the selection of Line 2167-9/2160-154 S-2. Approximately 70 plants were regenerated from Line 2167-9/2160-154 S-1. and either self-pollinated or cross-pollinated to the inbred maize lines A188. A619, A641, A661, A665, B37, B73. R806. and W153R.

B. Selection of Maize Cell Line 2167-9/2160-154 H-1

Line 2167-9/2160-154 H-1 was selected from maize cell culture using a similar protocol described in detail above except maize callus tissue was selected using the herbicide haloxyfop. Maize callus tissue was initially plated on 0.01 μM haloxyfop. At two-week intervals, surviving tissue was subcultured onto 0.05, 0.10 and 0.20 μM haloxyfop. Approximately 50 plants were regenerated from Line 2167-9/2160-154 H-1, and were self-pollinated.

C. Selection of Maize Cell Line 2167-9/2160-154 H-2

Line 2167-9/2160-154 H-2 was selected from maize cell culture using a similar protocol described in detail for line 2167-9/2160-154 H-1. No plants have been successfully regenerated from this line.

D. Characterization of Lines 2167-9/2160-154 S-1, H-1 and H-2

The tolerant callus cultures were characterized to determine the magnitude of sethoxydim and haloxyfop tolerance. Callus tissue from these lines was evaluated in experiments as described above in the characterization of line 2167-9/2160-154 S-2. Table II summarizes the results of these studies. Line 2167-9/2160-154 S-1 and Line 2167-9/2160-154 H-2 showed a four-fold increase in haloxyfop tolerance, while Line 2167-9/2160-154 H-1 exhibited approximately a 60-fold increase in haloxyfop tolerance. Neither haloxyfop selected line showed a significant degree of sethoxydim tolerance, while the sethoxydim selected line S-1 exhibited approximately a 100-fold increase in sethoxydim tolerance.

TABLE II Herbicide Tolerance of Cell Lines S-1, H-1 and H-2 Herbicide Cell Line Haloxyfop Sethoxydim 2167-9/2160-154 S-1  4¹ 100 2167-9/2160-154 H-1 61 0 2167-9/2160-154 H-2  4 0 ¹The numbers represent the fold increase in herbicide concentration that results in a 50% reduction in growth of the selected cell lines compared to the unselected control cell line 2167-9/2160-154.

E. Herbicide Inhibition of Acetyl Coenzyme A Carboxylase of Maize Cell Lines S-1, H-1 and H-2

Acetyl Coenzyme A Carboxylase (ACCase) was extracted from cell lines S-1, H-1 and H-2 and assayed as described in detail for maize cell line S-2, above. Table III below summarizes the results of these studies. The ACCase from line S-1 was more tolerant of both sethoxydim and haloxyfop, while the ACCase from line H-1 was more tolerant of haloxyfop, but not of sethoxydim. The ACCase from line H-2 showed no difference from the unselected parent line 2167-9/2160-154 in sensitivity to either herbicide.

However, cell line H-2 exhibited approximately a five-fold higher level of ACCase activity as compared to the unselected parent line 2167-9/21 60-154. Thus, selection for sethoxydim or haloxyfop tolerance resulted in a less sensitive ACCase in cell line S-1 and H-1. as well as a higher level of ACCase activity in cell line H-2.

TABLE III Herbicide Inhibition of ACCase of Maize Cell Lines S-1, H-1 and H-2 Herbicide Cell Line Haloxyfop Sethoxydim 2167-9/2160-154 S-1 3 4 2167-9/2160-154 H-1 7 0 2167-9/2160-154 H-2 0 0 ¹The numbers represent the fold increase in herbicide concentration that inhibits ACCase activity of the selected cell lines by 50% compared to the unselected parent cell line 2167-9/2160-154.

Deposit of Seeds

Seeds from representative S-2 plants (Ex. III (B)) and H-1 plants (Ex. IV(B)) have been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 USA on October 25. 1988 and assigned accession numbers ATCC 40507, and ATCC 40508, respectively.

EXAMPLE V Formation of cDNA Clones Encoding ACCase

A. ACCase Purification

The acetyl CoA carboxylase enzyme was isolated and purified from plant tissues and characterized. The purified enzyme was used to generate antibody reagents useful in identifying cDNA clones encoding the gene or portions of the gene for ACCase.

ACCase was extracted from frozen shoots of 7-d-old maize (Zea Mays L. inbred A619 or B73) seedlings grown in a growth chamber (24° C., 90% RH, 16-h daylength at 210 μE m⁻² s⁻¹). The outermost leaf and blade were removed and the remainder of the shoot was frozen in liquid N₂. Embryos and endosperm tissue from developing kernels were harvested from field-grown ears at 36 to 40 days after pollination (DAP). Black Mexican Sweet corn (BMS) maize suspension cells were obtained from cultures as previously described (W. B. Parker et al., Plant Physiol., 99, 1220-1225 (1990)). Tissues were stored in liquid N, until used.

Extraction and purification steps were performed at 0 to 4° C. Crude extracts of leaf, bundle sheath strands, embryo, endosperm, and BMS cells were prepared from frozen tissue as described by W. B. Parker et al., Proc. Nat'l Acad. Sci. USA, 87. 7175-7179 (1990), except that extraction buffer contained 0.1 M Tricine-KOH, pH 8.3, 0.3 M glycerol, 5 mM DTT. 2 mM Na₂EDTA, and 0.5 mM phenyl methonyl sulfonyl fluoride (PMSF). Triton X-100 (0.01% v/v) was added to bundle sheath strand extracts and to some whole leaf extracts. For some experiments, additional protease inhibitors (leupeptin, 2 μg mL; pepstatin A, 100 μg mL⁻¹; benzamidine, 1 mM; P-amino-n-caproic acid, 5 mM; and soybean trypsin inhibitor. 10 μg mL⁻¹) were included. Filtered homogenates were centrifuged 20 minutes at 30,000 g. A portion of the crude supernatant fraction was immediately boiled 5 minutes in 1 volume of SDS sample buffer (W. B. Parker et al., Plant Physiol., 92, 1220-1225 (1990)) for SDS-PAGE analysis; the remainder was desalted on a 10-mL Sephadex G-25 column into extraction buffer minus PMSF.

ACCase was purified from the crude extract supernatant in four steps. This fraction was brought to 30% saturation with solid (NH₄)₂SO₄, stirred 15 minutes, and centrifuged 20 minute at 20,000 g. The supernatant was then brought to 40% saturation with (NH₄)₂SO₄ solution, stirred 30 minutes, and centrifuged. The pellet was dissolved in 5 mL extraction buffer, microfuged 5 minutes, and the resulting supernatant was applied to a Sephacryl S-300 gel filtration column (Pharmacia; 2.5×46 cm) equilibrated with S-300 buffer (0.1 M Tricine-KOH, pH 8.3, 0.5 M glycerol, 0.5 mM DTT, 2 mM NaSO mM KCl). In later experiments a Sephacryl S-400 column was used. Fractions (2.5 mL) were eluted at 0.75 mL min⁻¹. ACCase activity eluted shortly after the void A₂₈₀ peak (V₀=75 mL). Active fractions were pooled, brought to 4.25 mM MgCl₂ (from a 0.5 M solution), and applied at 0.2 mL min⁻¹ to a Blue Sepharose CL-6B (Pharmacia; 1.5×15 cm) equilibrated with Blue sepharose buffer (S-300 buffer containing 4.25 mM MgCl₂ and 10 mM NaHCO₃). The column was washed overnight with 150 mL buffer (0.45 mL min⁻¹). ACCase activity was then eluted with 50 mL buffer plus 10 mM ATP (0.45 mL min⁻¹). Active fractions were pooled and applied to an FPLC Mono-Q HR 5/5 anion-exchange column (Pharmacia) equilibrated with S-300 buffer minus KCl. The column was washed with 30 mL S-300 buffer minus KCl and then with a 48-mL. 0 to 500 mM KCl gradient in S-300 buffer (0.25 mL min⁻¹). Fractions (1 mL) from the two peaks of ACCase activity were pooled separately. All purification fractions were. desalted into S-300 buffer and assayed for ACCase activity and protein.

ACCase was also analyzed from mesophyll chloroplasts and bundle sheath strands. Mesophyll chloroplasts from homogenates of 7- to 8-day-old seedlings that were kept in the dark 24 hours prior to harvesting were isolated on a linear Percoll gradient according to J. D. Burton et al., Pesticide Biochemistry and Physiology, 34, 76-85 (1989), except that buffers contained 0.6 M sorbitol and centrifugation g-forces were reduced by 25%. Intact chloroplasts were taken from the discrete lower green band present after Percoll gradient centrifugation (G. Morioux et al., Plant Physiol., 67, 470-473 (1981)). Pelleted chloroplasts were lysed by resuspending them in ACCase extraction buffer plus PMSF and 0.01% (v/v) Triton X-100. Bundle sheath strands were obtained from the original leaf homogenate material retained on a 70-μm filter after re-homogenizing the retentate five times in a total of 2 L buffer. Triton X-treated, desalted leaf, mesophyll chloroplast, and bundle sheath strand extracts were assayed for activities of Rubisco (G. Zhu et al., Plant Physiol., 97, 1348-1353 (1991)), NADP-dependent malate dehydrogenase (M. D. Hatch et al., Biochem. Biophys. Res. Commun., 34, 589-593 (1969)), phosphoenolpyruvate carboxylase (R. C. Leegood et al., “Isolation of Membranes and Organelles from Plant Cells,” Academic Press, New York, 185-210(1983)), catalase (Worthington Biochemicals, 1972), and fumarase (R. L. Hill et al., Methods Enzymol., 13, 91-99 (1969)), and for total chlorophyll (D. E. Arnon, Plant Physiol., 24, 1-5 (1949)). Mesophyll chloroplast preparations were judged to be relatively free of contamination by bundle sheath chloroplasts because they contained 3-fold greater NADP-dependent malate dehydrogenase and one-tenth as much Rubisco activity (mg-1 chlorophyll) than bundle sheath strand extracts. Mesophyll chloroplast preparations also contained ≦2.6% as much catalase, fumarase, and phosphoenolpyruvate carboxylase activities (mg⁻¹ chlorophyll) as did whole-leaf extracts, indicating they were relatively free of peroxisomal, mitochondrial, or cytoplasmic components.

ACCase activity as measured by acetyl-CoA-dependent H¹⁴CO (ICN. 2.07 GBq mmol⁻¹) incorporation into acid-stable product previously shown to be malonyl-CoA (J. D. Burton et al., Pesticide Biochemistry and Physiology, 34, 76-85 (1989)). Assays of desalted purification fractions or crude, desalted tissue extracts contained up to 50 and 25% (v/v) enzyme, respectively. In some experiments methylcrotonyl-CoA or propionyl-CoA were substituted for acetyl-CoA (E. S. Wurtele et al., Archives of Biochemistry and Biophysics, 278, 179-186 (1990)). Avidin (10 U mL⁻¹) was included in some assays. Herbicide inhibition assays contained 1% (v/v) ethanol plus or minus 1 μM haloxyfop (2-[4-[[3-chloro-5-(trifluoromethyl)-2-pyridinyl]oxy]phenoxy]propanoic acid, Dow Chemical Co. analytical grade racemic mixture) or 10 μM sethoxydim (2-[1[(ethoxylmino)butyl]-5-[2-(ethylthio)-propyl]-3-hydroxy-2-cyclohexene-1-one, Li salt, BASF Corp. technical grade). Data are means plus standard error of three assays.

Protein concentrations were determined in duplicate with the Bio-Rad Coomassie blue dye-binding assay as described by the manufacturer, using BSA as the standard.

Centrifuged crude extracts and proteins in purification fractions and immunoprecipitation supernatants were separated by SDS-PAGE in 6 or 7.5% gels as previously described (W. B. Parker et al., Plant Physiol., 92 1220-1225 (1990)). Purification fractions were precipitated in 10% (v/v) TCA, washed with 80% (v/v) acetone, and air-dried 10 minutes prior to electrophoresis. Proteins in gels were stained with silver (J. Heukeshoven et al., Electrophoresis, 6 103-112 (1985)). High molecular weight protein standards for SDS-PAGE (Pharmacia) were used to estimate polypeptide masses.

The four-step purification procedure shown in Table IV typically yielded 30 to 190 μg of highly purified ACCase from 50 grams (fresh weight) of maize inbred A619 or B73 seedling leaves. ACCase activity in the crude supernatant fraction precipitated between 30 and 40% saturation with (NH₄)₂SO₄, which appeared to increase total ACCase activity approximately 38%. Crude a extract components might have depressed the reaction rate shown in Table IV because the assay mixture contained 50% enzyme (v/v). In tests of fractions from another purification, enzyme velocity was proportional to enzyme concentration in assay mixtures containing up to 25% (v/v) crude extract, but 50% (v/v) mixtures were not tested. ACCase activity eluted from the Sephacryl S-300 gel filtration column slightly after the green void peak. Approximately 56% of the S-300 fraction ACCase activity was recovered from the Blue Sepharose column, primarily in the initial ATP-containing fractions (12.5 mL). Both 10 mM NaHCO₃ and 4.25 mM MgCl₂ (1- and 0.85-fold standard assay concentrations, respectively) were included in the Blue Sepharose buffer because they improved the total and specific ACCase activity remaining after batch absorption to Blue Sepharose beads, elution with ATP, and desalting into extraction buffer minus PMSF. Neither NaHCO₃ nor MgCl₂ improved enzyme stability of crude extracts. Mono-Q anion-exchange chromatography resulted in separation of two ACCase activity peaks which eluted at approximately 210 mM (designated ACCase II) and 250 mM KCl (designated ACCase I), as previously observed for a hybrid maize variety (J. L. Howard et al., FEBS Lett., 261, 261-264 (1990)). ACCase I comprised about 85% of the total activity recovered from the column (29% of the original crude extract activity) and had high specific activity (Table IV). The specific activity of ACCase II was less than 30% that of ACCase I. Both activities were inhibited >90% by avidin, as previously reported (J. L. Howard et al., FEBS Lett., 261, 261-264 (1990)). The mass of native ACCase I was estimated to be approximately 490 kD by gel filtration on Superose 6.

TABLE IV Purification of AC Case I From Maize Inbred A619 Seedling Leaves^(a) All fractions were desalted into S-300 buffer and assayed for protein and acetyl CoA dependent incorporation of [¹⁴C]HCO₃ ⁻ into acid-stable products Specific Fold Protein Activity Activity Purifi- Activity Step mg unitsb units/mg cation Yield Crude extract 215 2.45 0.0114 1 100 30-40% (NH₄)₂S0₄ 45.1 3.37 0.0748 6.56 138 S-300 10.7 3.35 0.313 27.5 137 Blue Sepharose 1.50 1.86 1.24 109 76 Mono-Q (ACCase I) 0.130 0.720 5.54 486 29 ^(a)Data are from one purification experiment starting with 50 g fresh weight of tissue and are representative of data obtained for eight purifications. ^(b)Unit = 1 μmol acid-stable product min⁻¹.

B. Formation and Specificity of Antibodies to ACCase

Antibodies are sensitive reagents that allow for the identification of gene products from cDNA and other cloned genes. Antibodies to purified ACCase were prepared and used to screen for cDNA clones encoding all or a portion of a gene for ACCase.

Antiserum to maize ACCase was obtained by immunizing a female New Zealand White rabbit (Egli et al., Plant Physiol., 101, 499 (1993)). An intramuscular injection of 100 μg of Mono-Q-purified, SDS-denatured ACCase I in Freund's complete adjuvant was followed by subcutaneous injections of 20 to 100 μg of gel-purified ACCase I polypeptide in acrylamide plus incomplete adjuvant every 4 to 6 weeks, for a total of six injections. Serum was stored at −20° C. in 0.02% (w/v) NaN₃.

For Western blots, proteins in SDS gels were electrophoretically transferred to Immobilon (W. B. Parker et al., Plant Physiol., 92, 1220-1225 (1990)) for 1 hour at 20 V in a Bio-Rad Transphor semi-dry blotter and then stained with Ponceau S (E. Harlow et al., “Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988)). Destained blots were blocked with Tris-buffered saline plus 0.5% (v/v) Tween-20 (Bio-Rad), and 10% (w/v) bovine serum (for antiserum blots only). ACCase and biotinylated proteins were detected with immune serum (1/10.000) plus goat anti-rabbit IgG-alkaline phosphatase conjugate or with avidin-alkaline phosphatase (W. B. Parker et al., Plant Physiol., 92, 1220-1225 (1990)). Blots were repeated at least three times.

For immunoprecipitations, equal ACCase activities (0.58 nmol min⁻¹) in crude extracts were desalted into S-300 buffer containing 0.1 M KCl and incubated 1 hour at 25° C. with 16 μL buffer or with 16 μL serum consisting of 0 to 100% ACCase antiserum in preimmune serum. Immune complexes were incubated 1 hour at 25° C. with a 2-fold (IgG binding) excess of Protein A-agarose and then microfuged 5 minutes to obtain immunoprecipitation supernatant fractions. ACCase activity of supernatants was expressed as a percent of the 100% preimmune serum control. Data are means plus SE of three replicate assays for each of two sets of extracts.

Western blots and silver-stained gels of purification fractions separated by 7.5% SDS-PAGE showed that neither ACCase I nor ACCase II Mono-Q fractions contained biotinylated polypeptides smaller than 212 kD. A polypeptide >212 kD was the primary protein component of the ACCase I Mono-Q fragment (FIG. 5). The ACCase II fraction contained a biotinylated polypeptide >212 kD and a large amount of a 55 kD non-biotinylated polypeptide. Fractions from earlier purification steps contained additional biotinylated proteins of approximately 74, 75, and 125 kD (FIG. 5).

To better compare the biotinylated polypeptides >212 kD in ACCase fractions I and II, we used 6% SDS-PAGE, which showed that the mass of ACCase II was approximately 8 kD less than that of ACCase I. Molecular masses were estimated to be 219 kD (ACCase II) and 227 kD (ACCase I), based on comparisons with polypeptide standards and the observation (N. R. Palosaari, Plant Physiol., 99(S), 359 (1992)) that, on Phastgels (Pharmacia), ACCase I polypeptide was slightly smaller than dodecameric horse spleen ferritin (238 kD; M. Heusterspreute et al., FEBS Lett., 129, 322-327 (1981)). All purification fractions through the Blue Sepharose step contained both ACCase I and II polypeptides. Rapid extraction of leaves in buffer containing five additional protease inhibitors, or a 4 hour incubation of extracts at 25° C., had little or no effect on the relative amounts of the two polypeptides, suggesting that ACCase II is not a breakdown product of ACCase I.

Antiserum to ACCase I strongly recognized the ACCase I polypeptide in crude extracts and showed little or no recognition of ACCase II polypeptides. No bands were recognized by preimmune serum. Assuming that avidin binds similarly to ACCase I and II polypeptides, it appears that the amount of ACCase II on the Western blot was slightly less than the amount of ACCase I. However, the relative staining with antibody compared to avidin indicated that the antibody had significantly less affinity for ACCase II than ACCase I.

To determine whether the same ACCase polypeptides were expressed in different maize cell types, proteins in mesophyll chloroplasts and crude extracts of leaves, endosperm tissue, embryos, and BMS cells were separated by SDS-PAGE. All preparations contained a predominant biotinylated polypeptide of approximately 227 kD (ACCase I) that was strongly recognized by ACCase antiserum or avidin. Similar 227 kD band densities were observed when gel lanes were probed with either avidin or ACCase antiserum. The 219 kD ACCase II polypeptide was readily detected in leaves only by avidin binding, but was in low abundance or not detected in extracts from other tissues. Only the 227 kD ACCase I polypeptide was detected in purified mesophyll chloroplasts, however, suggesting that the 219 kD ACCase II polypeptide is localized elsewhere in mesophyll cells or in other cell types of young leaves. ACCase activity and a >212 kD biotinylated polypeptide(s) were also found in bundle sheath strand extracts, but low yields prevented us from determining the type of ACCase present. Two other major biotinylated polypeptides of 75 and 74 kD were found in all tissues. Other non-biotinylated proteins of 66 kD (faint) and 55 kD were also recognized by ACCase antiserum. The 55 kD polypeptide was only found in leaves; it was also present in both ACCase I and II Mono Q fractions (FIG. 5) and was identified as the Rubisco large subunit based on its comigration with protein immunoprecipitated bN, spinach Rubisco antiserum.

ACCase antiserum immunoprecipitated at least 75% of ACCase activity from crude, desalted extracts of leaves endosperm tissue, embryos, and BMS cells (FIG. 6), indicating that most of the ACCase activity in these tissues is immunologically related to the ACCase I polypeptide of leaves. Less activity was precipitated from leaves (75%) than from other tissues, particularly embryos (98%). Compared to immunoprecipitation, inhibition of ACCase activity by antiserum in solution was less than 20% as effective in reducing ACCase activity.

The substrate specificity of ACCase from different purification fractions was examined to compare [¹⁴C]HCO₃-incorporation in the presence of different acyl-CoA substrates. Both ACCase I and II utilized propionyl-Co-A 40 to 50% as rapidly as acetyl-CoA at 50 to 500 μM substrate even though they contained no biotinylated polypeptides (FIG. 5) the size of known propionyl CoA carboxylases (70 to 75 kD; see E. S. Wurtele et al., Archives of Biochemistry and Biophysics, 278, 179-186 (1990)). Activities in the presence of both acetyl-CoA and propionyl-CoA (250 or 500 μM each) were approximately 90 (ACCase I) to 130% (ACCase II) that of 500 μM acetyl-CoA alone. Crude leaf extracts utilized propionyl-CoA and methylcrotonyl-CoA 60% as efficiently as acetyl-CoA. Methylcrotonyl CoA carboxylase activity was reduced 85% by gel filtration and was completely removed by Blue Sepharose affinity chromatography.

ACCase I and II differed significantly in their inhibition by either haloxyfop or sethoxydim (FIG. 7). Acetyl-CoA or propionyl-CoA-dependent H¹⁴CO₃- incorporation by ACCase I was strongly inhibited (65 to 80%) by 1 μM haloxyfop or 10 μM sethoxydim, while ACCase II activity was inhibited less than 50% for all herbicide/substrate combinations examined.

C. Cloning and Identification of Maize cDNA Clones Encoding ACCase

Maize cDNA clones encoding a portion of the ACCase game were identified by screening a DNA library generated from maize. The cDNA clones were used to identify the sequence of the ACCase gene and to identify the genomic DNA fragments encoding the gene or genes for ACCase.

A λgt11 cDNA library from maize inbred A188 seedlings was prepared by standard method for oligo-dT priming, as described for pea cDNA. (Gantt and Key, Eur. J. Biochem., 166:119-1125 (1987). Plaque lifts of the maize cDNA library were screened with maize ACCase antiserum (Egli et al., Plant Physiol., 101, 499 (1993)) to identify plaques expressing ACCase-like proteins as described by Sambrook et al., cited supra. (1989). The initial screen of 800,000 plaques yielded 120 positives. Rescreening and plaque purification reduced the number of positives to 14. All 14 clones bound ACCase antibodies that, when eluted from plaque lifts (J. Hammarback et al., J. Biol. Chem., 265:12763 (1990)), recognized a 227-kD biotinylated polypeptide on SDS-PAGE western blots of embryo and leaf crude extracts. The strongest western blot reaction was obtained with cDNA clone #15-14. The six best clones were digested with EcoRI to excise maize cDNA inserts. Total insert sizes ranged from 1.2 to 5.1 kb indicating the clones most likely did not contain the full coding sequences for the mature 219-kD and 227-kD ACCase polypeptides (minimum estimates of 6.1 and 6.3 kb, respectively).

Clone #15-14 contained three EcoRI fragments of 2.0, 1.2 and 0.23 kb shown in FIG. 8. Southern blots showed that the 1.2 and 2.0-kb fragments of clone #15-14 each hybridized to different fragments in the other five clones, with the exception of clone #4-4 which only contained a 1.2-kb fragment. The six maize cDNA clones contained EcoRI fragments that hybridized to a large transcript (ca. 7.8 kb) on Northern blots of total RNA from maize leaves, embryos and endosperm (FIG. 9). BMS cell culture RNA also contained a 7.8 kb transcript, but the hybridization signal is not evident on this exposure (FIG. 9). The relative abundance of the 7.8-kb transcript in embryos was higher than the other sources which is consistent with their ACCase activity.

The three EcoRI fragments were subcloned from cDNA clone #15-14 into BlueScript vector and sequenced by the dideoxy chain termination method (Sequenase 2.0 USB) initially using T3 and T7 primers and then oligonucleotide primers based on insert sequence. A clone #16-6 was also sequenced in a similar manner. Clone #16-6 included three EcoRI fragments of 3.1 kb, 1.2 kb, and 0.23 kb and had additional sequence located upstream from that of clone #15-14. After comparing the sequence and determining that the sequence was the same, the additional 1.2 kb sequence at the 5′ end was sequenced.

Clone #18-5 was sequenced in a similar manner. Clone #18-5 included 3.9 kb, 1.2 kb, and 0.23 kb EcoRI fragments and contains an additional 1.9 kb 5′ sequence upstream from clone #15-14. Subclone #18-5I (3.9 kb EcoRI fragment) has been deposited with the American Type Culture Collection and given Accession No. 69236.

GenBank, PIR-29, and Swiss-Prot 19 data banks have been searched for amino acid homology with the corresponding amino acid sequences of the three subclones of clone #18-5. Peptide sequences corresponding to the maize cDNA subclones had higher similarity to chicken, rat, and yeast ACCases than to any other peptide sequence in the data banks. FIG. 8 illustrates the relative organization of the 3.9, 1.2 and 0.23-kb EcoRI fragments of clone #18-5 and their co-linearity and extent of amino acid identity with chicken ACCase cDNA sequence. This comparison shows that the maize clone #18-5 has a large region near the 3′ end with high amino acid identity (40 to 61%) to chicken ACCase, a longer region with 23% identity in the middle of the 3.9-kb sequence, and a short region with 52% identify near the 5′ of the 3.9 kb sequence.

Portions of the sequence of the #18-5I subclone have been identified as encoding domains of the ACCase enzyme of functional significance. Those functional regions include a fragment that spans the presumed transcarboxylase active site in the enzyme having the following presumed sequence SEQ ID NO:2:

      1112-856 GTT CCT GCA AAC ATT GGT GGA CCT CTT CCT ATT ACC AAA CCT CTG GAC CCT CCA GAC AGA CCT GTT GCT TAC ATC CCT GAG AAC ACA TGC GAT CCA CGT GCA GCT ATC TGT GGT GTA GAT GAC AGC CAA GGG AAA TGG TTG GGT GGT ATG TTT GAC AAA GAC AGC TTT GTG GAG ACA TTT GAA GGA TGG GCA AAA ACA GTG GTT ACT GGC AGA GCA AAG CTT GGA GGA ATT CCT GTG GGC GTC ATA GCT GTG GAG ACA

This functional domain is contained in the sequence 1112 to 856 base pair from the 3′ stop codon or carboxy terminus region of the ACCase coding sequence of maize. This transcarboxylase active sequence is also present in clone #15-14.

Another functional region that has been identified spans the 12 base pair sequence encoding the biotin binding site having the following peptide sequence SEQ ID NO:3:

5′ GTT ATG AAG ATG 3′     Val Met Lys Met

The biotin binding site is encoded approximately 30% in from the 5′ (N-terminus) end of rat, chicken and yeast ACCase genes. These functional domains are useful in mapping and further identifying other cDNA and/or genomic fragments encoding ACCase genes.

The cDNA clones encoding portions of the acetyl CoA carboxylase genes are useful to identify the sequence of the gene or genes and are useful as probes to locate the genomic copies of the gene or genes. Because the ACCase antibodies used to screen the λgt11 library recognize both the 219 and 227 kD ACCase polypeptides, it has not been determined which polypeptide is encoded by these less than full length clones. It is likely that the majority of the clones encode the 227 kD polypeptide since that polypeptide is more abundant in the leaf tissue source of the DNA library and the antibodies have a higher affinity for the 227 kD ACCase polypeptide.

EXAMPLE VI Isolation and Sequencing of Genomic Encoded ACCase Genes and a Complete cDNA Sequence of a Maize ACCase Gene

The maize genome has been analyzed to identify copy number and location of the genomic copies of ACCase gene or genes. Four distinct types of maize ACCase genomic clones have been identified, termed A1, A2, B1 and B2 (see below).

To obtain genomic copies of ACCase genes, a maize B73 genomic library (Clontech, Palo Alto, Calif.) was screened with the 2 kb subclone from #15-14 and several clones of about 15 kb were identified as having homology to the ACCase cDNA. Restriction mapping and partial sequence analysis revealed two types of genomic clones (Type A and Type B) that differed in restriction sites and in their position relative to the ACCase partial cDNA sequence as shown in FIG. 8.

The 2.5 kb EcoRI-SalI fragment (#16) from the Type A genomic clone and the 3.0 kb EcoRI-EcoRI fragment (#34) from the Type B genomic clone were shown to hybridize to the 3.9 kb probe from #18-5 and were subcloned into the Bluescript vector and sequenced. Approximately 1.5 kb of DNA sequence from the genomic Type A 2.5 kb fragment were 100% identical to coding sequence from the 3.9 kb cDNA subclone #18-5I described in Example V; the remaining sequence exhibited no identity with the cDNA clone and presumably represents a noncoding intron sequence. A 350 nucleotide sequence derived from the genomic Type B 3.0 kb fragment was about 95% identical to the cDNA clone indicating that its coding sequence differs from that of genomic Type A. These results also indicate that the maize genome encodes at least two different genes encoding a polypeptide having acetyl CoA carboxylase activity.

To identify and clone the remainder of the gene representing the amino-terminus of maize ACCase, additional regions from the Type A genomic clone have been subcloned and partly sequenced. The 3.5 kb HindIII—HindIII fragment (#28) has been sequenced for about 400 nucleotides from each end. The 3′ end of #28 shows significant homology to the amino acid sequence from the chicken sequence located about 0.5 kb from the start of the chicken gene.

The complete sequence for fragment #28 can be obtained and analyzed to determine whether it contains the 5′ end of the ACCase coding region. The start of the transcribed region, and thus the likely start of the coding region for ACCase, can be identified by using the genomic clones in RNAse protection analysis (J. Sambrook et al., “Molecular Cloning—A Laboratory Manual,” Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)). Based on sequence data from the genomic clone, alignment, as shown in FIG. 8, with sequences of other ACCases and identification of potential open reading frames, oligonucleotide primers can be constructed to synthesize cDNA molecules representing the amino terminus of the ACCase gene. These molecules can be hybridized to genomic Type A DNA fragments such as #28 and the nonhybridizing portions digested with S1 nuclease. The end of the protected fragment are determined by analysis on a DNA sequencing gel.

To synthesize the remaining coding region between the end of the cDNA clone #18-5 and the start of transcription, two oligonucleotide primers were synthesized. Primer I is complementary to the DNA sequence: (SEQ ID NO:4)

5′ GCCAGATTCC ACCAAAGCAT ATATCC 3′

near the 5′ end of cDNA subclone #18-5I and can be used as a primer for synthesis of cDNA molecules from maize seedling, leaf or embryo RNA.

A primer corresponding to a DNA sequence near the transcription start site can be used in combination with primer 1 for the amplification of DNA by the polymerase chain reaction (PCR). Several independent clones are then sequenced and their sequences compared to the known sequence of the Type A genomic clone to determine the exact coding sequence corresponding to that maize gene for ACCase. A similar strategy can be used to obtain the complete coding sequence for genomic Type B ACCase.

The remaining cDNA sequence was obtained by three successive rounds of RT-PCR using oligonucleotide primers based on genomic apparent exon (5′) and known cDNA (3′) sequences. The primers used to amplify nucleotides 1-240 of the cDNA were 28sst-a5+ (SEQ ID NO. 7) and 28sst-6at3+ (SEQ ID NO:8), nucleotides 217-610 of the cDNA were 28sst-5+(SEQ ID NO:9) and 28-2t3+(SEQ ID NO:10) and nucleotides 537-2094 of the cDNA were ACCPCR5′ (SEQ ID NO:11) and 155-(SEQ ID NO:4) (Table V). PCR products corresponding to nucleotides 1-240, 217-610. and 537-2094 of the final sequence were cloned into PCR-script (Stratagene).

TABLE V cDNA 5′ primer 5′ primer 3′ primer 3′ primer Position designation sequence designation sequence nt-1-240 28sst-a5+ GGTCTTCAATTGTGCTGTCTGG 28sst-6at3+ CCTTGACGAACAGACTGGCTGTGC (SEQ ID NO:7) (SEQ ID NO:8) nt 217-610 28sst-5+ CACAGCCAGTCTGTTCGTCAAGG 28-2t3+ CCTCTACGTAATTGGTCAGC (SEQ ID NO:9) (SEQ ID NO: 10) nt 537-2094 ACCPCR5′ CATAGCTATGGCAACTCCGG I55 GGATATATGCTTTGGTGGAATCTGGC (SEQ ID NO:11) (SEQ ID NO:4)

The original 5.4-kb cDNA clone #18-5 and PCR products from at least three individual PCR per oligonucleotide pair were sequenced in both directions by the dideoxy chain-termination method, using either Sequenase II (U.S. Biochemicals) or ABI 373 (Applied Biosystems, Inc.) protocols. No sequence differences were found in regions of clone overlaps. The complete sequence of the cDNA of maize ACCase (nucleotides 1-7470 SEQ ID NO:5) and its corresponding amino acid sequence (amino acids 1-2325 SEQ ID NO:6) are shown in FIGS. 13 and 14. The 7470 bp cDNA includes a 459 nucleotide 3′ untranslated region and 36 nucleotides of 5′ untranslated sequences.

The first Met codon in the cDNA (nucleotides 37-39) was identified as the start codon based on its similarity to consensus initiation sequences (Kozak, J. Cell. Biol., 108, 229 (1989); Lutcke et al., Embo. J., 6, 43 (1987)). An in-frame stop was found in the genomic sequence 6 nucleotides upstream of the sequenced cDNA, and RT-PCR analysis of this region suggested that the in frame stop codon was also present in the cDNA. The 3′ end of the coding sequence was defined by two stop codons present in the large open reading frame after nucleotide 7011. The translated coding sequence predicted a polypeptide of 2325 amino acids (257 kD; SEQ ID NO:6) which was 79 to 81% identical to the multifunctional (MF) ACCases from alfalfa (Shorrosh et al., Proc. Nat'l. Acad. Sci., 91, 4323 (1994)) and wheat (Gornicki et al. Proc. Nat'l Acad. Sci., 91, 6860 (1994)), and to a 118-amino acid predicted polypeptide of a rice expressed sequence tag (Genbank accession #D39099, T. Sasaki), but only 53 to 55% identical to ACCase from other eukaryotes.

In a pileup alignment of plant ACCases (Genetics Computer Group. Madison, Wis.), Met I of both maize and Brassica napus ACCases was located about 130 amino acids upstream of the conserved sequence VDEFCKALGG, compared to only 25 amino acids upstream for other plant ACCases. The predicted 2325 amino acids of maize ACCase contains a biotinylation site at position 806, within the conserved MKM motif (Ton et al., Eur. J. Biochem., 215, 687 (1993)). The arrangement and amino acid sequence of binding sites (Shorrosh et al., Proc. Nat'l. Acad. Sci., 91, 4323 (1994)) for ATP (amino acids 318-333), biotin (amino acids 799-811; biotin at 806), acetyl-CoA (amino acids 1952-1961), and carboxybiotin (amino acids 1662-1711) were highly conserved among all MF ACCases.

EXAMPLE VII Characterization of other Genomic Clones

The initial restriction fragment length polymorphism (RFLP) analysis of EcoRI-digested total DNA from three maize inbred lines showed one band when probed with the 2 kb subclone from #15-14 (internal to gene) and two bands when probed with the 1.2 kb subclone (near the 3′ end of the gene). Fragments homologous to the 2 kb probe were monomorphic and the more intense of the two bands hybridizing with the 1.2 kb probe was dimorphic. As discussed in Example V, these results support the view that maize contains at least two distinguishable ACCase genes and that they may be quite similar for much of the coding region. Additional genomic Southern blots of a set of recombinant inbred lines were used to map polymorphisms for the ACCase probes to maize chromosomes. One polymorphism was mapped to the short arm of chromosome 2: other polymorphisms were not evident in these initial tests to identify a chromosomal location for other maize ACCase genes.

The isolation and restriction mapping of additional genomic clones from a B73 genomic library (Clontech) resulted in the identification of four different types of clones termed A1, A2, B1 and B2 (FIGS. 16-19) which had 96% nucleotide sequence identity. Types A and B correspond to previously published pA3 and pA4 cDNAs (Ashton et al., Plant Mol. Biol., 24, 35 (1994)) and differ from pA3 and pA4 by 4% in their coding sequences.

Type A and B genomic clones have linear sequence homology except for an insertion in an intron of the Type B genes about 1400 bp 3 ′ of the A1 (SEQ ID NO:5) translation start site. Analysis of the insert boundaries revealed a 3-bp target site duplication and a 6-bp direct repeat, and further sequence analysis showed the presence of two new and unique LINE elements (Long Interspersed Nuclear Elements) in B1 and B2. Mammalian LINE elements are highly abundant (10⁴ to 10⁵ copies), 6 to 7 kb long, and have frequent 5 ′-end deletions and an A-rich 3′ terminus. They are flanked by short direct repeats, and contain two ORFs, one encoding a reverse transcriptase. Three LINE elements (Cin4, 50-100 copies in maize; del2, 250,000 copies in lily; BNR1, 2-5% of genome in sugarbeet) have been described in plants (Leeton et al., Mol. Gen. Geneti., 237, 97 (1993); Schmidt et al., Chromo. Res., 3, 335 (1995); Schwarz-Sommer et al., EMBO J., 6, 3873 (1987)). Maize ACCase B1 has one unique LINE element and B2 has two. The two B2 LINE elements were characterized by differences in their reverse transcriptase sequence. The B genomic clone inserts have characteristic LINE features including cysteine motifs and a possible polyA tail, and high abundance. The LINE insert also has been found in an intron of the maize Shrunken-2 gene (Hannah et al., Plant Physiol., 98, 1214 (1992)).

The partial nucleotide sequence (3489 nucleotide) of a Type A1 ACCase genomic clone is shown in FIG. 16 (SEQ ID NO:12). The clone is a HindIII fragment which includes nucleotides 1-931 of the cDNA in FIG. 13 (SEQ ID NO:5), and the first four introns within the coding region, at positions 240 (460 nucleotides), 296 (480 nucleotides), and 872 (76 nucleotides) of SEQ ID NO:5. The clone also has 1395 nucleotides 5′ to the cDNA of SEQ ID NO:5 (i.e., 1431 nucleotides 5′ of the translational start at nucleotide position 1432).

The partial nucleotide sequence (1328 nucleotide) of another Type A clone is shown in FIG. 17 (SEQ ID NO:1 ). The partial sequence is all 5′ untranslated sequence and contains a 7 base insert between nucleotides 279-290, but is otherwise identical to SEQ ID NO:12.

The partial nucleotide sequence of six Type A2 clones is shown in FIG. 18 (1565, 1168, 638, 558, 976 and 852 nucleotides; SEQ ID NOs 14, 15, 16, 17. 18 and 19, respectively).

Within the A1--A2 clone pair, identified differences are in introns and 5′ UTR sequences. The A2 genomic clone is weakly amplified with Type A1 PCR primers specific for the 5′ UTR if the 3′ primer employed is for a conserved amino acid sequence found in all ACCases (e.g., 28sst-110F, ACTGTGCGTTTGAGAAGGTC, SEQ ID NO:23, and 28sst-2T3+, CCTCTACGTAATTGGTCAGC, SEQ ID NO:24). The A2 amplified product is the same size as that from the Type A1 genomic clone, and restriction analysis indicated a difference in sequence from A1. Sequence differences in the 5′ region should provide a means to distinguish between expression of A1 and A2 ACCase genes and to determine whether A2 also encodes a CTP.

The partial nucleotide sequence (231, 207 and 180 nucleotides) of three Type B clones is shown in FIG. 19 (SEQ ID NOs 20. 21 and 22, respectively).

The cDNAs corresponding to genomic clones A2, B1 and B2 are cloned and sequenced in a manner similar to that described above. The derived amino acid sequences are aligned with known ACCase sequences. If putative CTP sequences are identified, functionality is tested as described below. Also if the tissue specificity and developmental timing of expression differ for different ACCase genes, the sequences of the promoter regions of the corresponding genomic clones are compared. Gene-specific probes for specific ACCase genes can provide more information on their roles in lipid synthesis (plastid and cytoplasmic isoforms), secondary metabolism (cytoplasmic isoforms), and herbicide resistance (likely plastid isoforms).

A 3′ Type A1 ACCase cDNA probe mapped to chromosome 2S (Egli et al., Maize Genetics Newsletter, 68, 92 (1994)) and to 10 L (Caffrey et al., Maize Gen. Coop., 69, 3 (1995)). Two 5′ Type A1 cDNA probes which span the transit peptide mapped to chromosome 2S in the same location as the 3′ probe (see maize genetic map, 1996 version, Maize Genomic Database). PCR primers 28sst-97F (CCTTTTTATGGCACTGTGCG, SEQ ID NO:25) and 28sst-6t3+; (CATCGTAGCCTATATGAGGACG, SEQ ID NO:26) located in non-coding regions of A1 that span the chloroplast transit peptide were used to amplify a B73 chromosome-specific product which segregated with the resistance trait. A nearby 5′ primer (28sst-a5+, SEQ ID NO:7) amplified all genotypes and functioned as a positive control. Herbicide resistance due to the Acc1-S3 mutation segregates (29/29 individuals to date) with production of a Type A 5′ end-specific PCR product derived from the mutant parent while herbicide sensitive plants lack the transit sequence (15/17 progeny). Two individual plants which contained B73-specific DNA at this location died of unknown causes while grown in the presence of herbicide.

Mutations in maize that confer resistance to cyclohexanedione and aryloxyphenoxypropionate herbicides by means of an altered ACCase target are found at two non-allelic loci, Acc1 and Acc2. A1 and A2 appear to encode plastidic ACCases and correspond to the Acc1 and Acc2 herbicide resistance loci. Acc2 has been mapped to 10 L (VanDee, M. S. Thesis, University of Minnesota (1994)). Acc1 is the site of five allelic mutations including Acc1-S2 and -S3 (Marshall et al., Theor. Appl. Genet., 83, 435 (1992)), and has been mapped to chromosome 2.

Only one plastidic ACCase polypeptide was identified by SDS-PAGE of maize leaf extracts, although 2-D gel analyses might provide evidence for a second, highly similar isoform. Of the two ACCase isoforms, only ACCase I shows altered herbicide inhibition in Acc1-S2 mutants, and most of thc ACCase activity in leaves and developing embryos is herbicide-resistant and thus attributed to the Acc1-S2 gene product.

Although a 3′ ACCase probe has been mapped both to 10 L near Acc2-S5 and to 2S, the conserved sequence of ACCase genes and lack of polymorphism in multiple bands complicates identification of genes encoded at these loci. The Type A1 ACCase gene is probably located on chromosome 2, since (i) 5′ untranslated and chloroplast transit peptide probes from Type A1 hybridize to two bands (dark and light) in maize inbreds, and (ii) analysis of maize-oat addition lines carrying maize chromosomes 2 through 9 indicates the dark band is on chromosome 2 and the light band is on chromosome 1 or 10.

Type B ACCase genes are likely to encode cytosolic isoforms. Given that cytosolic malonyl-CoA is a precursor in the synthesis of many secondary metabolites including flavonoids (e.g., maysin, a corn silk component associated with corn earworm resistance), these cytosolic ACCases can have agronomic utility.

Northern blot analysis of total maize RNA with an ACCase probe (nucleotides 3400-5932) showed a single 8.3 kilobase band. To determine whether the expression of ACCase RNAs was developmentally regulated, blots of total RNA from 16 to 42 DAP (days after pollination) embryos were probed with an ACCase cDNA fragment. Transcript abundance peaked about 23 DAP and the steady state pattern was similar to in vitro ACCase enzyme activities and protein measured from developing embryos. Type A- and B- specific ³²P-CTP-labeled antisense transcripts were 780 nt long (662 nt of ACCase sequence+118 nt of vector/promoter sequence) and were identical except for 15 base mismatches scattered along their length. Each antisense transcript was hybridized to total RNA from embryos at 16, 20, 23, and 42 DAP and digested with RNAse A/TI mixture to yield a 662-base fragment specific to the probe used. The results showed that the Type A transcript was more abundant than Type B at all tested stages, and that only Type A remained high in older embryos. Types A and B had similar expression patterns and peaked around 20-23 DAP. The ratio of Type A:B mRNA in leaves was about 2: 1, similar to its relative abundance in cDNA expression libraries.

EXAMPLE VIII Expression of the Maize ACCase Chloroplast Transit Peptide

The N-terminus of the predicted maize ACCase polypeptide is longer than that of predicted cytosolic ACCase isoforms and has several characteristics typical of chloroplast transit peptides within the first approximately 73 amino acids of the predicted N-terminal sequence. The CTP cleavage site motif is not found in the putative maize ACCase CTP, although only about 30% of known CTPs contain this consensus sequence (Gavel and von Hejne, FEBS Lett., 261, 455 (1990)). However, the maize ACCase N-terminus appears to have several other properties typical of known CTPs: (1) a lack of acidic residues in amino acids 1-49, (2) high Ser+Thr content (69% within amino acid residues 23-35), (3) an R-rich region between S- and D-rich regions in amino acid residues 36-49, and (4) a predicted turn→β sheet within amino acid residues 58-73 (von Hejne and Nishikawa, FEBS Lett., 278, 1(1991)).

The ability of the amino acid sequence contained within the N-terminal 100 amino acids of the translated maize acetyl-CoA carboxylase (ACCase) cDNA to direct the N-terminal portion of the maize ACCase biotin carboxylase domain into chloroplasts was tested in vitro by methods used extensively in the literature (see Cline et al., J. Biol. Chem., 260, 3691 (1985); Lubben and Keegstra, Proc. Nat'l. Acad. Sci., 83, 5502 (1986)). The criteria for import was that (1) in vitro-synthesized, ³⁵S-labeled protein was imported into chloroplasts, and (2) the transported protein was smaller than the original translation product, by an amount which corresponds to the removal of the expected CTP. Import studies utilized either maize or pea chloroplasts. Pea chloroplasts have been reported to correctly import proteins from many different species, including maize (Nieto-Sotelo et al. Plant Physiol., 93. 1321 (1990)). Alternatively, the function of the putative maize ACCase CTP is tested by inserting the first 258 coding nucleotides of maize ACCase in frame with and 5′ of a GUS reporter gene in pBI221 (Clontech). This construct and the pBAR plasmid are used to co-transform maize “Black Mexican Sweet” suspension cells by particle bombardment. Basta-resistant transformants are selectedm and GUS activity and/or protein is assayed in surviving cultures or in plasmids isolated from transformants.

A partial ACCase construct consisting of nucleotides 1-833 of SEQ ID NO:5 including the putative CTP (nucleotides 37 to 256) and the first domain within the biotin carboxylase region (identified by amino acid sequence comparison with E. coli biotin carboxylase; see Waldrop et al. Biochem., 33, 6249 (1994)) was amplified by PCR and cloned into the EcoRV site of PCR-script (Stratagene) to create the plasmid pBCN1. A corresponding plasmid lacking CTP sequences (nucleotides 278-833) was also made (-pBCN1). The protein encoded by -pBCN1 begins at amino acid residue 83 (Val—Met).

Constructs were transformed into E. coli SURE cells (Stratagene).

Restriction analysis of pBCN1 with BamHI and HindIII indicated that the 5′ end of the ACCase was located adjacent to the T7 RNA polymerase binding site in PCR-Script. A partial sequence of pBCN1 obtained by using the T7 sequencing primer and the ABI373 automated sequencing protocol confirmed this orientation and showed that the pBCN1 insert sequence was identical to maize ACCase cDNA for at least the first 300 nucleotides and that it included the maize ACCase Met 1 ATG. An acyl carrier protein clone containing a CTP (spinach ACPII, a gift of Dr. John Ohlrogge, Michigan State University) can be used as a positive control. These constructs are used for in vitro transcription, translation, chloroplast import, and SDS-PAGE analysis of products in the same manner as pBCN1.

Purified pBCN1 was digested with EcoRI to linearize the plasmid at the 3′ end of the BCN1 insert, electrophoresed in 1.5% agarose, and the plasmid band at approximately 3.8 kb was excised and Gene-Cleaned (BioLab 101). The purified band was digested with 20 μg proteinase K to remove any residual RNAse, extracted with phenol and then chloroform under RNAse-free conditions. DNA content was estimated by ethidium bromide fluorescence in droplets, relative to λDNA standards (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (1989). One pg of pBCN1 DNA was transcribed into capped RNA with the T7/mMessage mMachine kit (Ambion). Uncapped transcripts (Sp6 RNA polymerase; Promega) of pea RUBISCO small subunit (SSU; Anderson et al., Biochem. J., 240, 709 (1986)) were also transcribed. RNA yield was estimated by determining the % incorporation of a ³²P-ATP (Amersham) into a precipitable product, according to the Ambion kit instructions. Electrophoresis and autoradiography of ³²P-labeled product showed that it contained a single RNA band of approximately 895 nucleotides, as expected.

The RNA transcripts were translated into ³⁵S-labeled polypeptides with Ambion's wheat germ IVT kit and approximately 45 μCi ³⁵S-methionine (Amersham; 37 TBq/mmol) in a 50-μL reaction. Labeled proteins were held on ice 6 hours prior to their use in chloroplast import experiments.

Pea (cv. “Little Marvel”) and maize (inbred A188) plants were grown in a growth chamber at 25° C., 16 hour day length. Chloroplasts were isolated from pea and maize leaves 7 days after planting, respectively, as previously described (Burton et al., Pestic Biochem. and Physiol., 34, 76 (1989); Egli et al., Plant Physiol., 101, 499 (1993)). Intact mesophyll chloroplasts were washed in resuspension buffer [50 mM HEPES-KOH, pH 7.8 plus 0.33 M (pea) or 0.66M (maize) sorbitol] in preparation for import assays. Suspensions were diluted to obtain 75 μg chlorophyll/0.3 ml (Arnon, Plant Physiol., 24, 1 (1949)).

Import experiments were carried out essentially as described by Cline et al.(J. Biol. Chem., 260, 3691 (1985)). Import reactions containing 0.3 ml pea or maize chloroplast suspension, 40 μl ³⁵S-translation mixture, 3 mM Mg-ATP and 10 mM Met were incubated under light for 1-30 minutes at 25° C.

Unimported proteins were digested for 30 minutes with 40 μg of thermolysin, and proteolysis was stopped with 10 mM EDTA.

Chloroplasts were re-isolated by centrifuging them through I-ml 40% v/v Percoll gradients in the presence of resuspension buffer plus 3 mM Mg-ATP, 10 mM Met, and 20 mM EDTA, washed twice in the same buffer, and resuspended in 65 μl of 1 mM MgCl₂/10 mM Tris buffer, pH 8.0. Chloroplasts were lysed by three cycles of freeze-thawing in liquid N₂, microfuged, and aliquots of the supernatants and of the original in vitro-translated proteins were analyzed by SDS-PAGE in 8-25% gradient Phast gels (Pharmacia), followed by direct detection of radiolabeled proteins in the wet gels (AMBIS) (FIG. 20).

As estimated by SDS-PAGE, a 30 minute import converted the original 32-kD BCN1 polypeptide to a doublet of 27.2 and 27.5 kD in maize and produced an additional 30-kD band in pea (FIG. 20A). Neither maize or pea chloroplasts imported -pBCN1-derived polypeptides. Formation of the 27.2-kD polypeptide likely resulted from cleavage after amino acid #47-49, a likely cleavage site because it lies between S- and D-rich regions, and R residues are located at -2,-7, and -8 (S. Gavel and G. Von Heijne, FEBS Lett. 261, 455 (1990)).

Time-dependence of BCN1 import was further examined (1-30 minutes) (FIG. 20B) to determine if any imported polypeptides were a result of incomplete processing or proteolysis. Import was maximal after 15 minutes, but import time had no effect on the relative amounts of different-sized import products. Higher amounts of ATP (5 mM) stimulated import relative to lower amounts of ATP (<0.2 mM). The data suggest that, in maize, efficient cleavage of BCN1 occurs at two closely adjacent sites and that partially processed products are also formed during BCN1 import by pea chloroplasts. Therefore, nucleotides 1-833 of the maize ACCase gene encode a CTP.

EXAMPLE IX Expression of a cDNA Clone or Genomic Clones Encoding the ACCase Gene

The cDNA and genomic clones encoding all or a portion of the ACCase gene can be subcloned into a known expression system and the gene products reactive with the antibodies specific for maize ACCase can be identified using a Western blot. For example, the ACCase cDNA clones are inserted into two transformation plasmids: (i) Glb1exp which contains the embryo-specific maize Globulin1 (Glb1) promoter and 3′ regions (Belanger et al., Genetics, 129, 863 (1991)); and (ii) pAHC17 which contains the maize ubiquitin (Ubi-1) constitutive promoter and first exon and intron, and the NOS 3′ terminator (Christensen et al., Plant Mol. Biol., 18, 675 (1992); Toki et al., Plant Physiol, 100, 1503 (1992)). The 3′ end of the A₁ cDNA has a unique Sal/I site just 3 ′ of the stop codon which is used to ligate into a Sal/I site in both plasmids ahead of the construct terminator. Other cloning sites will be added as needed to the plasmids or cDNA to complete the ligation of the 5′ end. The gene products can also be further characterized structurally and/or enzymatically. This will ensure that the genomic and cDNA clones that encode acetyl CoA carboxylase can be screened for promoters that provide for overproduction of the native or herbicide tolerant ACCase enzyme in plants.

For example, the 2 kb EcoRI fragment from clone #15-14 can be subcloned into a plant transformation plasmid pBI121 or pBI221 downstream from the 35S CaMV promoter and upstream from the nopaline 3′ polyadenylation signal sequence, as described in Jefferson, Plant Molec. Biol. Reptr., 5, 387-405 (1987). This plasmid can then be used to transform plant cells such as tobacco, Brassica and Arabidopsis cells using protoplast or biolistic transformation, as described by W. J. Gordon-Kamm et al., Plant Cell, 2, 603-618 (1990); M. E. Fromm et al., Bio/Technology, 8, 833-839 (1990); An, Methods in Enzymology, 153, 292 (1987); and D'Hafluin, The Plant Cell, 4, 1495 (1992). An increase in transient expression can be detected using quantitative Western blotting with antibodies specific for the ACCase enzymes. Polyclonal antibodies to maize ACCase most likely do not substantially crossreact with ACCase from dicots like tobacco or Arabidopsis.

Alternatively, the ACCase gene can be subcloned along with the 35S CaMV promoter into a binary Ti vector pGA482, as described in An, cited supra., which is a binary Ti vector system and can be used to transform plant cells by Agrobacterium-mediated transformation. Stably transformed plants can be generated by standard methods as described in Example III, and levels of expression of ACCase genes can be determined by quantitative Western blots, as described in Harlow and Lane, Antibodies, Cold Spring Harbor Laboratories (1988). The ability to monitor expression of cloned ACCase genes will permit the identification of promoters that provide for enhanced expression of the ACCase gene. The expression system can be used to screen for those promoters that enhance gene expression of the ACCase gene at least about 5 to 10-fold over the endogenous levels of ACCase produced normally in the plant cells. Because the 35S CaMV promoter is known as a strong promoter, it is likely this promoter will provide for at least a 5-fold increase in the expression of ACCase over that normally produced in the plant cell.

In addition, this expression system can be used to screen antisense DNA sequences. For example, an antisense sequence can be obtained that is complementary to an about 0.5 kb region of the maize ACCase cDNA that has high homology with a portion of the chicken ACCase gene and contains the sequence for the presumed transcarboxylase active site domain, as shown in FIG. 8. The antisense sequence could be subcloned into a pBI121 or pBI221 expression under the control of an inducible plant promoter, such as nitrite reductase promoter (Back et al., Plant Molec. Biol., 17:9-18 (1991)). The ability of the antisense sequence to inhibit expression of the native ACCase gene can be evaluated in transformed cells, for example as described in Hamilton et al., Nature, 346:284-287 (1990).

EXAMPLE X Identification and Cloning of the Gene From Herbicide Resistant Maize Cell Lines

Herbicide resistant maize cell lines were generated as described in Examples I, II, and IV. These herbicide resistant cell lines have been shown to produce an ACCase enzyme that is less sensitive to inhibition by sethoxydim or haloxyfop. The genes encoding the herbicide resistant forms of the ACCase will be identified and cloned using standard methods as described in Sambrook et al., Guide to Molecular Cloning: A Laboratory Manual (1989). The genes encoding the herbicide resistant forms of ACCase can then be introduced into herbicide sensitive plant species by standard methods to confer herbicide resistance. For example, the ACCase enzyme in the maize cell line 2167-9/2160-154 S-1 is at least 100-fold less sensitive to sethoxydim than the wild-type.

DNA from the cell line or plants will be obtained and digested with EcoRI and/or other appropriate restriction enzymes, according to standard methods. The restriction enzyme digest will be separated out by agarose gel electrophoresis and probed with either the 2 kb or the 3.9 kb cDNA ACCase probe described in Example V. Fragments hybridizing to the 2 kb or 3.9 kb probe will be subcloned into a Bluescript vector and portions of the gene will be sequenced, as described in Example V, to verify that the entire ACCase gene has been isolated.

To confirm that the clone encodes the ACCase gene, it will be subcloned into the pBI121 or pBI221 expression vector, as described in Example VIII. The ACCase gene product expressed by the clone in either Black Mexican sweet corn cells or tobacco cells will be evaluated for reactivity with ACCase specific antibodies, by enzyme activity, and/or resistance of the enzyme activity to inhibition with sethoxydim and/or haloxyfop. It is likely that the cloned gene will encode an ACCase which is resistant to inhibition by sethoxydim and haloxyfop. This gene can then be introduced into an herbicide-sensitive embryogenic plant cell or an embryo, including maize cells or immature embryos, to confer herbicide resistance to that plant species upon regeneration.

The complete coding sequence encoding the herbicide resistant form of the ACCase enzyme will be cloned into a plant transformation vector such as pBI121 or pBI221 as described in Jefferson, Plant Molec. Biol. Reporter, 5:387-405 (1987). This vector contains the 35S CaMV constitutive promoter, the β-glucuronidase structural gene, and the nopaline synthase 3′ polyadenylation signals. The β-glucuronidase gene is replaced with a cloned ACCase gene. Optionally, the cloned ACCase gene can be combined with natural or synthetically produced chloroplast transit peptide sequence from pea, as described in Keegstra & Olsen, Ann. Rev. Plant. Physiol./Mol. Biol. 40:471-501 (1989) and/or unique restriction sites introduced so the cloned gene can be distinguished from the endogenous maize ACCase gene. Standard methods of subcloning will be utilized as described in Sambrook et al., cited supra.

For transformation of maize cells, type II calli can be transformed using biolistic transformation, as described by W. J. Gordon-Kamm et al., Plant Cell, 2, 603-618 (1980); M. E. Fromm et al., Bio/Technology, 8, 833-839 (1990), and D. A. Walters et al., Plant Molecular Biology, 18, 189-200 (1992). Alternatively, type I embryogenic calli can be transformed using electroporation after mechanically or enzymatically wounding calli, as described by D'Hafluin et al., The Plant Cell, 4:1495 (1992). Once the cloned gene is introduced into these cells and transformants are selected, typically by antibiotic resistance, fertile transgenic maize plants can be regenerated, as described by D'Hafluin et al., cited supra. Fertile transgenic plants can be evaluated for herbicide tolerance, as described in Example III. It is likely that the fertile transgenic plants having and expressing a cloned ACCase gene as an ACCase resistant to sethoxydim and/or haloxyfop will exhibit herbicide tolerance as compared to the corresponding untransformed plant.

EXAMPLE XI Generation of Transgenic Plants Having an Increase in Oil Content

Once identified and cloned, the gene or genes from maize acetyl CoA carboxylase can be introduced into monocot or dicot plant species, including maize, under the control of a promoter that provides for overexpression of the ACCase enzyme. The overexpression of the ACCase enzyme is likely to lead to an increase in the oil content of the plants and seeds.

Naturally occurring soybeans that have a high oil content and soybeans that have a low oil content have been identified. The acetyl CoA carboxylase from both types of soybeans was isolated, as described in Example V. The activity of the enzyme was measured as a function of the time of seed development and the results are shown in FIG. 11.

The results in the FIG. 11 indicate that higher oil content soybean is associated with a 2-fold increase in the ACCase activity during early to mid stages of development when compared with a low oil content soybean. Thus, increased expression of the ACCase gene correlates with an increase in the oil content of the seed. Total oil content of the seed was also measured at maturity (60 days). The high oil producing cell lines, Anoka and PI28C. 134, have a total oil content of 21.8% and 19.9%, respectively. In contrast, the low oil soybean line of M76-395, has an oil content of 13.6% oil. Thus, the increase of ACCase expression early in seed development correlates with a higher total oil content in the seed at maturity.

A gene encoding a genomic maize acetyl CoA carboxylase can be isolated, as described in Example V, and used to transform plant species by protoplast or biolistic transformation. If the (gene is combined with a strong promoter, such as the 35S cauliflower mosaic virus promoter, overexpression of the ACCase gene is likely. Alternatively, selecting transformed cells with multiple copies of the gene can also result in transformed cells overexpressing the ACCase gene. The gene can be cloned into a vector such as pBI21 or pBI221, as described by Jefferson, cited supra. This vector contains the 35S cauliflower mosaic virus promoter, the β-glucuronidase structural gene, and the nopaline synthase 3′ polyadenylation signals. The cloned ACCase gene can replace the β-glucuronidase gene and then be used to transform plant cells, including maize, as described in Example VIII.

Transformed cells can be screened for overproduction of ACCase. The presence of the cloned gene can be verified by identifying the unique restriction enzyme sites incorporated into the cloned gene. ACCase levels can be assessed by standard enzyme assay methods and quantitative Western blots using antibodies specific for maize ACCase. Fatty acid and lipid content in cells lines overproducing ACCase are likely to be elevated and can be assessed using standard methodologies, as described in Clark & Snyder, JACS, 66:1316 (1989). Transformed cell lines overproducing ACCase and having increased total oil content will be used to regenerate fertile transgenic plants and seeds, as described in D'Hafluin, cited supra.

EXAMPLE XII Generation of Transgenic Plants Having an Increase in Plastidic ACCase Activity

Maize embryos are transformed with sense and antisense cDNA constructs encoding the plastidic A1 ACCase. Selected transgenic cultures and regenerated transgenic plants and progeny are subjected to detailed analyses of: ACCase transcript levels: activity of ACCase I (plastidic) and ACCase II (presumably cytosolic) in various plant tissues; fatty acid synthesis; lipid accumulation in kernels (primarily embryos); and other plant traits. The culmination of these tests indicates whether plastidic ACCase activity can be modified via transformation and whether fatty acid synthesis is affected.

1. Transformation of maize embryos and plant regeneration

Immature maize embryos of the Hi-II genotype are transformed by particle bombardment according to slight modifications from previously described procedures (Fromm et al., Biotech., 8, 833 (1990); Koziel et al., Biotech., 11, 194 (1993)). This procedure has been employed in transformation studies for bombardment of approximately 15,000 embryos which were then selected for Basta-resistant callus (bar selectable marker gene expression), and regenerated into plants. Transformed (Basta-resistant) plants are obtained from 1-2% of the initial embryos and, when separate plasmids are used for co-transformation, the nonselected transgene is recovered in about 50% of the Basta-resistant plants.

Basta-resistant, hemizygous transformed (T₀=F1) plants will be tested by PCR or Southern blots for the presence of A1 ACCase sequences unique to the transformation vector, grown to maturity in growth chambers, greenhouse or field, and self-pollinated when possible, or backcrossed to a nontransformed parent. F2 or backcross progeny are grown in the greenhouse and field and tested for Basta resistance and presence of the A1 ACCase transgene to identify homozygous transgenic plants. Homozygous inbred transgenic lines are then developed.

2. Analysis of ACCase A1 transformants

Plants recovered from at least 100 independent transformation events (i.e., from different bombarded embryos) for both the UBI1 and GLB1 vectors are recovered. Regenerated plants are tested for the presence of the intact ACCase A1 transgene and its cosegregation with the Basta-resistance marker. Homozygous and heterozygous transgenic lines are assayed for total ACCase activity in leaves for UBI1 transformants and in developing embryos (22-26 DAP) for both UBI1 and GLB1 transformants. Sethoxydim and haloxyfop inhibition are used to quickly determine the levels of herbicide-sensitive ACCase I (plastidic) and the herbicide-insensitive ACCase II activity in both leaves and embryos. Increased expression of the ACCase A1 transgene contributes to plastidic ACCase activity and not to ACCase II activity. Kernel fatty acid and oil content are analyzed at maturity and their relationship with ACCase I determined by methods well known to the art.

Transformants that differ in kernel ACCase activity and/or fatty acid and oil content are then selected for more detailed analysis of embryos throughout development (4-day intervals from 16 DAP to maturity). These analyses include RNAse protection assays to determine total A1+A2 transcript levels using a non-specific probe and to determine relative levels of endogenous A1 versus transgene A1 transcripts by use of antisense riboprobes spanning the 5′ UTR region of the A1 transgene constructs. Western blots of total proteins separated by SDS-PAGE gels are probed with the ACCase I-specific antibody described hereinabove or with avidin and analyzed by densitometry to distinguish changes in the 227-kD ACCase I and 219-kD ACCase II isoforms. ACCase I activity, fatty acid and lipid content are determined in embryos at each stage of development. These analyses determine whether expression of an additional gene(s) for plastidic ACCase increases ACCase activity and consequently fatty acid and oil content in maize tissues, especially in embryos.

3. Transformation with maize ACCase A1 antisense

transformation vectors

Antisense transformation vectors were constructed by blunt-end ligation of nucleotides 1-833 of SEQ ID NO:5 in reverse orientation into multicloning sites of both the GLB1 and UBI1 plasmids. A sense construct with the same 833-bp cDNA sequence also was made with the GLB1 plasmid to serve as a transformation control. Insert orientations were verified by restriction mapping. UBI1 antisense, GLB1 antisense, and GLB1 sense constructs were introduced into >2100, >2900 and >2000 embryos, respectively, and Basta-resistant callus were selected.

If antisense expression results in significant reduction in ACCase activity, it may not be possible to obtain viable callus or plants from the constitutive UBI1 antisense transformants. Similarly, plants transformed with the embryo-specific GLB1 antisense construct may exhibit deleterious effects on embryo development. Thus, failure to obtain transgenic progeny containing the antisense ACCase gene from these transformations may indicate that ACCase activity cannot be downregulated without loss of viability.

4. Analysis of ACCase A1 antisense transformants

All Basta-resistant cultures will be regenerated. The presence of the UBI1 and GLB1 antisense constructs will be determined by PCR analysis for unique transgene sequences such as the Ubi-1 intron/ACCase A1 junction or Glb15′ UTR/ACCase A1 junction, or by Southern blotting to detect unique fragments. Plants and lines homozygous or heterozygous for the antisense transgene are analyzed for steady state level of the ACCase A1 antisense transcripts in appropriate tissues/organs (such as leaves, tassels, ears, embryos and endosperm for UBI1; leaves and embryos for GLB1) by using ACCase A1 sense riboprobes for hybridization on RNA blots or for RNAse protection assays. Total ACCase activity (both ACCase I and ACCase II isoforms) and fatty acid and lipid content are determined for the antisense transgenic lines and for corresponding tissues from nontransformed control plants. These analyses show whether ACCase A1 antisense transgenes are expressed in plants and, if so, whether expression is associated with reduced ACCase activity and altered fatty acid and lipid content in maize.

EXAMPLE XIII Expression of Plastidic and Cytosolic ACCases during Plant Development

Intact embryos are isolated from developing kernels of field-grown inbred B73 at 2 to 4 day intervals between 16-42 DAP and frozen immediately in liquid nitrogen. Samples also are saved for fresh and dry weight determinations. Subsamples from each stage are analyzed for total lipid and fatty acid content. Seedling leaves are sampled along the leaf blade ranging from the etiolated, meristematic basal region to the fully expanded, green tip. Leaves and other tissues (e.g., epidermis) of maize genotypes that accumulate anthocyanin pigments are also analyzed to assess whether a specific ACCase (such as a cytosolic ACCase) is more highly expressed in tissues in which malonyl-CoA also is required as a substrate for chalcone synthase in the flavonoid pathway leading to anthocyanin synthesis.

Gene-specific antisense riboprobes in RNAse protection assays are employed to determine A1, A2, B1 and B2 transcript levels. The corresponding sense transcripts are produced in vitro and used as standards to verify specificity and quantitate the sample transcript levels. Quantitation is done on an AMBIS radioanalytic image system. Herbicide inhibition of total ACCase activity provides an assessment of levels of herbicide-sensitive ACCase I (plastidic) and the herbicide-insensitive ACCase II activity in these tissues. ACCase I and II isoforms are separated by ion-exchange chromatography. Total proteins are separated by SDS-PAGE and Western blots probed with avidin to detect the biotinylated 227-kD ACCase I and 219-kD ACCase II isoforms or probed with ACCase I-specific antibodies.

While the present invention has been described in connection with the preferred embodiment thereof, it will be understood many modifications will be readily apparent to those skilled in the art, and this application is intended to cover any adaptations or variations thereof. It is manifestly intended this invention be limited only by the claims and equivalents thereof.

26 2001 base pairs nucleic acid single linear cDNA NO NO unknown 1 AGAGATGAAG CTCGCATGCC AATGCGCCAC ACATTCCTCT GGTTGGATGA CAAGAGTTGT 60 TATGAAGAAG AGCAGATTCT CCGGCATGTG GAGCCTCCCC TCTCTACACT TCTTGAATTG 120 GATAAGTTGA AGGTGAAAGG ATACAATGAA ATGAAGTATA CTCCTTCGCG TGACCGCCAA 180 TGGCATATCT ACACACTAAG AAATACTGAA AACCCCAAAA TGTTGCATAG GGTGTTTTTC 240 CGAACTATTG TCAGGCAACC CAATGCAGGC AACAAGTTTA GATCGGCTCA GATCAGCGAC 300 GCNAAGGTAG GATGTCCCGA AGAATCTCTT TCATTTACAT CAAATAGCAT CTTAAGATCA 360 TTGATGACTG CTATTGAAGA ATTAGAGCTT CATGCAATTA GGACAGGTCA TTCTCACATG 420 TATTTGTGCA TACTGAAAGA GCAAAAGCTT CTTGACCTCA TTCCATTTTC AGGGAGTACA 480 ATTGTTGATG TTGGCCAAGA TGAAGCTACC GCTTGTTCAC TTTTAAAATC AATGGCTTTG 540 AAGATACATG AGCTTGTTGG TGCAAGGATG CATCATCTGT CTGTATGCCA GTGGGAGGTG 600 AAACTCAAGT TGGACTGTGA TGGCCCTGCA AGTGGTACCT GGAGAGTTGT AACTACAAAT 660 GTTACTGGTC ACACCTGCAC CATTGATATA TACCGAGAAG TGGAGGAAAT AGAATCACAG 720 AAGTTAGTGT ACCATTCAGC CAGTTCGTCA GCTGGACCAT TGCATGGTGT TGCACTGAAT 780 AATCCATATC AACCTTTGAG TGTGATTGAT CTAAAGCGCT GCTCTGCTAG GAACAACAGA 840 ACAACATATT GCTATGATTT TCCGCTGGCC TTTGAAACTG CACTGCAGAA GTCATGGCAG 900 TCCAATGGCT CTACTGTTTC TGAAGGCAAT GAAAATAGTA AATCCTACGT GAAGGCAACT 960 GAGCTAGTGT TTGCTGAAAA ACATGGGTCC TGGGGCACTC CTATAATTCC GATGGAACGC 1020 CCTGCTGGGC TCAACGACAT TGGTATGGTC GCTTGGATCA TGGAGATGTC AACACCTGAA 1080 TTTCCCAATG GCAGGCAGAT TATTGTTGTA GCAAATGATA TCACTTTCAG AGCTGGATCA 1140 TTTGGCCCAA GGGAAGATGC ATTTTTTGAA ACTGTCACTA ACCTGGCTTG CGAAAGGAAA 1200 CTTCCTCTTA TATACTTGGC AGCAAACTCT GGTGCTAGGA TTGGCATAGC TGATGAAGTA 1260 AAATCTTGCT TCCGTGTTGG ATGGTCTGAC GAAGGCAGTC CTGAACGAGG GTTTCAGTAC 1320 ATCTATCTGA CTGAAGAAGA CTATGCTCGC ATTAGCTCTT CTGTTATAGC ACATAAGCTG 1380 GAGCTAGATA GTGGTGAAAT TAGGTGGATT ATTGACTCTG TTGTGGGCAA GGAGGATGGG 1440 CTTGGTGTCG AGAACATACA TGGAAGTGCT GCTATTGCCA GTGCTTATTC TAGGGCATAT 1500 GAGGAGACAT TTACACTTAC ATTTGTGACT GGGCGGACTG TAGGAATAGG AGCTTATCTT 1560 GCTCGACTTG GTATACGGTG CATACAGCGT CTTGACCAGC CTATTATTTT AACAGGGTTT 1620 TCTGCCCTGA ACAAGCTCCT TGGGCGGGAA GTGTACAGCT CCCACATGCA GCTTGGTGGT 1680 CCTAAGATCA TGGCGACCAA TGGTGTTGTC CACCTCACTG TTCCAGATGT CCTTGAAGGT 1740 GTTTCCAATA TATTGAGGTG GCTCAGCTAT GTTCCTGCAA ACATTGGTGG ACCTCTTCCT 1800 ATTACCAAAC CTCTGGACCC TCCAGACAGA CCTGTTGCTT ACATCCCTGA GAACACATGC 1860 GATCCACGTG CAGCTATCTG TGGTGTAGAT GACAGCCAAG GGAAATGGTT GGGTGGTATG 1920 TTTGACAAAG ACAGCTTTGT GGAGACATTT GAAGGATGGG CAAAAACAGT GGTTACTGGC 1980 AGAGCAAAGC TTGGAGGAAT T 2001 258 base pairs nucleic acid single linear cDNA NO NO unknown 2 GTTCCTGCAA ACATTGGTGG ACCTCTTCCT ATTACCAAAC CTCTGGACCC TCCAGACAGA 60 CCTGTTGCTT ACATCCCTGA GAACACATGC GATCCACGTG CAGCTATCTG TGGTGTAGAT 120 GACAGCCAAG GGAAATGGTT GGGTGGTATG TTTGACAAAG ACAGCTTTGT GGAGACATTT 180 GAAGGATGGG CAAAAACAGT GGTTACTGGC AGAGCAAAGC TTGGAGGAAT TCCTGTGGGC 240 GTCATAGCTG TGGAGACA 258 4 amino acids amino acid single linear peptide NO NO internal unknown 3 Val Met Lys Met 1 26 base pairs nucleic acid single linear cDNA NO NO unknown 4 GCCAGATTCC ACCAAAGCAT ATATCC 26 7470 base pairs nucleic acid single linear cDNA NO NO unknown 5 GGTCTTCAAT TGTGCTGTCT GGGCCACGGA ACGACAATGT CACAGCTTGG ATTAGCCGCA 60 GCTGCCTCAA AGGCCTTGCC ACTACTCCCT AATCGCCAGA GAAGTTCAGC TGGGACTACA 120 TTCTCATCAT CTTCATTATC GAGGCCCTTA AACAGAAGGA AAAGCCATAC TCGTTCACTC 180 CGTGATGGCG GAGATGGGGT ATCAGATGCC AAAAAGCACA GCCAGTCTGT TCGTCAAGGT 240 CTTGCTGGCA TTATCGACCT CCCAAGTGAG GCACCTTCCG AAGTGGATAT TTCACATGGA 300 TCTGAGGATC CTAGGGGGCC AACAGATTCT TATCAAATGA ATGGGATTAT CAATGAAACA 360 CATAATGGAA GACATGCCTC AGTGTCCAAG GTTGTTGAAT TTTGTGCGGC ACTAGGTGGC 420 AAAACACCAA TTCACAGTAT ATTAGTGGCC AACAATGGAA TGGCAGCAGC AAAATTTATG 480 AGGAGTGTCC GGACATGGGC TAATGATACT TTTGGATCTG AGAAGGCAAT TCAACTCATA 540 GCTATGGCAA CTCCGGAAGA CATGAGGATA AATGCAGAAC ACATTAGAAT TGCTGACCAA 600 TTCGTAGAGG TGCCTGGTGG AACAAACAAT AATAACTACG CCAATGTTCA ACTCATAGTG 660 GGGATGGCAC AAAAACTAGG TGTTTCTGCT GTTTGGCCTG GTTGGGGTCA TGCTTCTGAG 720 AATCCTGAAC TGCCAGATGC ATTGACCGCA AAAGGGATCG TTTTTCTTGG CCCACCTGCA 780 TCATCAATGA ATGCTTTGGG AGATAAGGTC GGCTCAGCTC TCATTGCTCA AGCAGCCGGG 840 GTCCCAACTC TTGCTTGGAG TGGATCACAT GTTGAAGTTC CATTAGAGTG CTGCTTAGAC 900 GCGATACCTG AGGAGATGTA TAGAAAAGCT TGCGTTACTA CCACAGAGGA AGCAGTTGCA 960 AGTTGTCAAG TGGTTGGTTA TCCTGCCATG ATTAAGGCAT CCTGGGGAGG TGGTGGTAAA 1020 GGAATAAGAA AGGTTCATAA TGATGATGAG GTTAGAGCGC TGTTTAAGCA AGTACAAGGT 1080 GAAGTCCCTG GCTCCCCAAT ATTTGTCATG AGGCTTGCAT CCCAGAGTCG GCATCTTGAA 1140 GTTCAGTTGC TTTGTGATCA ATATGGTAAT GTAGCAGCAC TTCACAGTCG TGATTGCAGT 1200 GTGCAACGGC GACACCAGAA GATTATTGAA GAAGGTCCAG TTACTGTTGC TCCTCGTGAG 1260 ACAGTTAAAG CACTTGAGCA GGCAGCAAGG AGGCTTGCTA AGGCTGTGGG TTATGTTGGT 1320 GCTGCTACTG TTGAGTATCT TTACAGCATG GAAACTGGAG ACTACTATTT TCTGGAACTT 1380 AATCCCCGAC TACAGGTTGA GCATCCAGTC ACTGAGTGGA TAGCTGAAGT GAATCTGCCT 1440 GCAGCTCAAG TTGCTGTTGG AATGGGCATA CCTCTTTGGC AGATTCCAGA AATCAGACGT 1500 TTCTATGGAA TGGACTATGG AGGAGGGTAT GACATTTGGA GGAAAACAGC AGCTCTTGCT 1560 ACACCATTTA ATTTTGATGA AGTAGATTCT CAATGGCCAA AGGGCCATTG TGTAGCAGTT 1620 AGAATTACTA GTGAGGACCC AGATGATGGT TTCAAACCTA CTGGTGGGAA AGTGAAGGAG 1680 ATAAGTTTTA AAAGCAAGCC TAATGTTTGG GCCTACTTCT CAGTAAAGTC TGGTGGAGGC 1740 ATTCATGAAT TTGCTGATTC TCAGTTTGGA CATGCTTTTG CATATGGACT CTCTAGACCA 1800 GCAGCTATAA CAAACATGTC TCTTGCATTA AAAGAGATTC AGATTCGTGG AGAAATTCAT 1860 TCAAATGTTG ATTACACAGT TGACCTCTTA AACGCTTCAG ACTTCAGAGA AAACAAGATC 1920 CACACTGGTT GGCTGGATAC AAGAATAGCT ATGCGTGTTC AAGCTGAGAG GCCCCCATGG 1980 TATATCTCAG TGGTTGGAGG TGCTTTATAT AAAACAGTAA CCACCAATGC AGCCACTGTT 2040 TCTGAATATG TTAGTTATCT CACCAAGGGC CATATTCCAC CAAAGCATAT ATCCCTTGTC 2100 AATTCTACAG TTAATTTGAA TATAGAAGGG AGCAAATACA CAATTGAAAC TGTAAGGACT 2160 GGACATGGTA GCTACAGGTT GAGAATGAAT GATTCAACAG TTGAAGCGAA TGTACAATCT 2220 TTATGTGATG GTGGCCTCTT AATGCAGTTG GATGGAAACA GCCATGTAAT TTATGCAGAA 2280 GAAGAAGCTG GTGGTACACG GCTTCAGATT GATGGAAAGA CATGTTTATT GCAGAATGAC 2340 CATGATCCAT CGAAGTTATT AGCTGAGACA CCCTGCAAAC TTCTTCGTTT CTTGGTTGCT 2400 GATGGTGCTC ATGTTGATGC GGATGTACCA TACGCGGAAG TTGAGGTTAT GAAGATGTGC 2460 ATGCCTCTCT TGTCACCTGC TTCTGGTGTC ATTCATTGTA TGATGTCTGA GGGCCAGGCA 2520 TTGCAGGCTG GTGATCTTAT AGCAAGGTTG GATCTTGATG ACCCTTCTGC TGTGAAAAGA 2580 GCTGAGCCAT TTGATGGAAT ATTTCCACAA ATGGAGCTCC CTGTTGCTGT CTCTAGTCAA 2640 GTACACAAAA GATATGCTGC AAGTTTGAAT GCTGCTCGAA TGGTCCTTGC AGGATATGAG 2700 CACAATATTA ATGAAGTCGT TCAAGATTTG GTATGCTGCC TGGACAACCC TGAGCTTCCT 2760 TTCCTACAGT GGGATGAACT TATGTCTGTT CTAGCAACGA GGCTTCCAAG AAATCTCAAG 2820 AGTGAGTTAG AGGATAAATA CAAGGAATAC AAGTTGAATT TTTACCATGG AAAAAACGAG 2880 GACTTTCCAT CCAAGTTGCT AAGAGACATC ATTGAGGAAA ATCTTTCTTA TGGTTCAGAG 2940 AAGGAAAAGG CTACAAATGA GAGGCTTGTT GAGCCTCTTA TGAACCTACT GAAGTCATAT 3000 GAGGGTGGGA GAGAGAGCCA TGCACATTTT GTTGTCAAGT CTCTTTTCGA GGAGTATCTT 3060 ACAGTGGAAG AACTTTTTAG TGATGGCATT CAGTCTGACG TGATTGAAAC ATTGCGGCAT 3120 CAGCACAGTA AAGACCTGCA GAAGGTTGTA GACATTGTGT TGTCTCACCA GGGTGTGAGG 3180 AACAAAGCTA AGCTTGTAAC GGCACTTATG GAAAAGCTGG TTTATCCAAA TCCTGGTGGT 3240 TACAGGGATC TGTTAGTTCG CTTTTCTTCC CTCAATCATA AAAGATATTA TAAGTTGGCC 3300 CTTAAAGCAA GTGAACTTCT TGAACAAACC AAACTAAGTG AACTCCGTGC AAGCGTTGCA 3360 AGAAGCCTTT CGGATCTGGG GATGCATAAG GGAGAAATGA GTATTAAGGA TAACATGGAA 3420 GATTTAGTCT CTGCCCCATT ACCTGTTGAA GATGCTCTGA TTTCTTTGTT TGATTACAGT 3480 GATCGAACTG TTCAGCAGAA AGTGATTGAG ACATACATAT CACGATTGTA CCAGCCTCAT 3540 CTTGTAAAGG ATAGCATCCA AATGAAATTC AAGGAATCTG GTGCTATTAC TTTTTGGGAA 3600 TTTTATGAAG GGCATGTTGA TACTAGAAAT GGACATGGGG CTATTATTGG TGGGAAGCGA 3660 TGGGGTGCCA TGGTCGTTCT CAAATCACTT GAATCTGCGT CAACAGCCAT TGTGGCTGCA 3720 TTAAAGGATT CGGCACAGTT CAACAGCTCT GAGGGCAACA TGATGCACAT TGCATTATTG 3780 AGTGCTGAAA ATGAAAGTAA TATAAGTGGA ATAAGCAGTG ATGATCAAGC TCAACATAAG 3840 ATGGAAAAGC TTAGCAAGAT ACTGAAGGAT ACTAGCGTTG CAAGTGATCT CCAAGCTGCT 3900 GGTTTGAAGG TTATAAGTTG CATTGTTCAA AGAGATGAAG CTCGCATGCC AATGCGCCAC 3960 ACATTCCTCT GGTTGGATGA CAAGAGTTGT TATGAAGAAG AGCAGATTCT CCGGCATGTG 4020 GAGCCTCCCC TCTCTACACT TCTTGAATTG GATAAGTTGA AGGTGAAAGG ATACAATGAA 4080 ATGAAGTATA CTCCTTCGCG TGACCGCCAA TGGCATATCT ACACACTAAG AAATACTGAA 4140 AACCCCAAAA TGTTGCATAG GGTGTTTTTC CGAACTATTG TCAGGCAACC CAATGCAGGC 4200 AACAAGTTTA GATCGGCTCA GATCAGCGAC GCTGAGGTAG GATGTCCCGA AGAATCTCTT 4260 TCATTTACAT CAAATAGCAT CTTAAGATCA TTGATGACTG CTATTGAAGA ATTAGAGCTT 4320 CATGCAATTA GGACAGGTGA TTCTCACATG TATTTGTGCA TACTGAAAGA GCAAAAGCTT 4380 CTTGACCTCA TTCCATTTTC AGGGAGTACA ATTGTTGATG TTGGCCAAGA TGAAGCTACC 4440 GCTTGTTCAC TTTTAAAATC AATGGCTTTG AAGATACATG AGCTTGTTGG TGCAAGGATG 4500 CATCATCTGT CTGTATGCCA GTGGGAGGTG AAACTCAAGT TGGACTGTGA TGGCCCTGCA 4560 AGTGGTACCT GGAGAGTTGT AACTACAAAT GTTACTGGTC ACACCTGCAC CATTGATATA 4620 TACCGAGAAG TGGAGGAAAT AGAATCACAG AAGTTAGTGT ACCATTCAGC CAGTTCGTCA 4680 GCTGGACCAT TGCATGGTGT TGCACTGAAT AATCCATATC AACCTTTGAG TGTGATTGAT 4740 CTAAAGCGCT GCTCTGCTAG GAACAACAGA ACAACATATT GCTATGATTT TCCGCTGGCC 4800 TTTGAAACTG CACTGCAGAA GTCATGGCAG TCCAATGGCT CTACTGTTTC TGAAGGCAAT 4860 GAAAATAGTA AATCCTACGT GAAGGCAACT GAGCTAGTGT TTGCTGAAAA ACATGGGTCC 4920 TGGGGCACTC CTATAATTCC GATGGAACGC CCTGCTGGGC TCAACGACAT TGGTATGGTC 4980 GCTTGGATCA TGGAGATGTC AACACCTGAA TTTCCCAATG GCAGGCAGAT TATTGTTGTA 5040 GCAAATGATA TCACTTTCAG AGCTGGATCA TTTGGCCCAA GGGAAGATGC ATTTTTTGAA 5100 ACTGTCACTA ACCTGGCTTG CGAAAGGAAA CTTCCTCTTA TATACTTGGC AGCAAACTCT 5160 GGTGCTAGGA TTGGCATAGC TGATGAAGTA AAATCTTGCT TCCGTGTTGG ATGGTCTGAC 5220 GAAGGCAGTC CTGAACGAGG GTTTCAGTAC ATCTATCTGA CTGAAGAAGA CTATGCTCGC 5280 ATTAGCTCTT CTGTTATAGC ACATAAGCTG GAGCTAGATA GTGGTGAAAT TAGGTGGATT 5340 ATTGACTCTG TTGTGGGCAA GGAGGATGGG CTTGGTGTCG AGAACATACA TGGAAGTGCT 5400 GCTATTGCCA GTGCTTATTC TAGGGCATAT GAGGAGACAT TTACACTTAC ATTTGTGACT 5460 GGGCGGACTG TAGGAATAGG AGCTTATCTT GCTCGACTTG GTATACGGTG CATACAGCGT 5520 CTTGACCAGC CTATTATTTT AACAGGGTTT TCTGCCCTGA ACAAGCTCCT TGGGCGGGAA 5580 GTGTACAGCT CCCACATGCA GCTTGGTGGT CCTAAGATCA TGGCGACCAA TGGTGTTGTC 5640 CACCTCACTG TTCCAGATGT CCTTGAAGGT GTTTCCAATA TATTGAGGTG GCTCAGCTAT 5700 GTTCCTGCAA ACATTGGTGG ACCTCTTCCT ATTACCAAAC CTCTGGACCC TCCAGACAGA 5760 CCTGTTGCTT ACATCCCTGA GAACACATGC GATCCACGTG CAGCTATCTG TGGTGTAGAT 5820 GACAGCCAAG GGAAATGGTT GGGTGGTATG TTTGACAAAG ACAGCTTTGT GGAGACATTT 5880 GAAGGATGGG CAAAAACAGT GGTTACTGGC AGAGCAAAGC TTGGAGGAAT TCCTGTGGGC 5940 GTCATAGCTG TGGAGACACA GACCATGATG CAGATCATCC CTGCTGATCC AGGTCAGCTT 6000 GATTCCCATG AGCGATCTGT CCCTCGTGCT GGACAAGTGT GGTTCCCAGA TTCTGCAACC 6060 AAGACCGCTC AGGCATTATT AGACTTCAAC CGTGAAGGAT TGCCTCTGTT CATCCTGGCT 6120 AATTGGAGAG GCTTCTCTGG TGGACAAAGA GATCTCTTTG AAGGAATTCT TCAGGCTGGG 6180 TCAACAATTG TCGAGAACCT TAGGACATAT AATCAGCCTG CTTTTGTGTA CATTCCTATG 6240 GCTGGAGAGC TTCGTGGAGG AGCTTGGGTT GTGGTCGATA GCAAAATAAA TCCAGACCGC 6300 ATTGAGTGTT ATGCTGAAAG GACTGCCAAA GGTAATGTTC TCGAACCTCA AGGGTTAATT 6360 GAAATCAAGT TCAGGTCAGA GGAACTCCAA GACTGTATGG GTAGGCTTGA CCCAGAGTTG 6420 ATAAATCTGA AAGCAAAACT CCAAGATGTA AATCATGGAA ATGGAAGTCT ACCAGACATA 6480 GAAGGGATTC GGAAGAGTAT AGAAGCACGT ACGAAACAGT TGCTGCCTTT ATATACCCAG 6540 ATTGCAATAC GGTTTGCTGA ATTGCATGAT ACTTCCCTAA GAATGGCAGC TAAAGGTGTG 6600 ATTAAGAAAG TTGTAGACTG GGAAGAATCA CGCTCGTTCT TCTATAAAAG GCTACGGAGG 6660 AGGATCGCAG AAGATGTTCT TGCAAAAGAA ATAAGGCAGA TAGTCGGTGA TAAATTTACG 6720 CACCAATTAG CAATGGAGCT CATCAAGGAA TGGTACCTTG CTTCTCAGGC CACAACAGGA 6780 AGCACTGGAT GGGATGACGA TGATGCTTTT GTTGCCTGGA AGGACAGTCC TGAAAACTAC 6840 AAGGGGCATA TCCAAAAGCT TAGGGCTCAA AAAGTGTCTC ATTCGCTCTC TGATCTTGCT 6900 GACTCCAGTT CAGATCTGCA AGCATTCTCG CAGGGTCTTT CTACGCTATT AGATAAGATG 6960 GATCCCTCTC AGAGAGCGAA GTTTGTTCAG GAAGTCAAGA AGGTCCTTGA TTGATGATAC 7020 CAACACATCC AACACAATGT GTGCATGTCA CATCTTTTTG TTCTAGTACA TACATAGAAG 7080 GATATTGCTT GGTCTTGATT GATCATGTCT GATTTAAGTC GACTATTATT TCTTGGAATT 7140 TTCTTTTGGA CCTGGTGCTA TGGTTGATGG ATGTATATTG GATATGTGCG TTCTGCCAGG 7200 TGTAAGCACA AAGGTTTAGA CARAMMRARA RCAAGAGCGA GTGAACCTGT TCTGGTTTTG 7260 CAGTGGTTCA GTAAGGCAGA AAGTTGTTAA ACCGTAGTTC TGAGATGTAT TACCAGTGNC 7320 GCCATGCTGT ACTTTTAGGG TGTATAATGC GGATACAAAT AAACAATTTA GCGGTTCATT 7380 AAAGTTTGAA CTCAAATAAC ATGTTCTTTG TAAGCATATG TACCGTACCT CTACGTGAAA 7440 TAAAGTTGTT GAATTAGCAT TCGAAAAAAA 7470 2325 amino acids amino acid single linear protein NO NO N-terminal and C-terminal (full length protein) unknown 6 Met Ser Gln Leu Gly Leu Ala Ala Ala Ala Ser Lys Ala Leu Pro Leu 1 5 10 15 Leu Pro Asn Arg Gln Arg Ser Ser Ala Gly Thr Thr Phe Ser Ser Ser 20 25 30 Ser Leu Ser Arg Pro Leu Asn Arg Arg Lys Ser His Thr Arg Ser Leu 35 40 45 Arg Asp Gly Gly Asp Gly Val Ser Asp Ala Lys Lys His Ser Gln Ser 50 55 60 Val Arg Gln Gly Leu Ala Gly Ile Ile Asp Leu Pro Ser Glu Ala Pro 65 70 75 80 Ser Glu Val Asp Ile Ser His Gly Ser Glu Asp Pro Arg Gly Pro Thr 85 90 95 Asp Ser Tyr Gln Met Asn Gly Ile Ile Asn Glu Thr His Asn Gly Arg 100 105 110 His Ala Ser Val Ser Lys Val Val Glu Phe Cys Ala Ala Leu Gly Gly 115 120 125 Lys Thr Pro Ile His Ser Ile Leu Val Ala Asn Asn Gly Met Ala Ala 130 135 140 Ala Lys Phe Met Arg Ser Val Arg Thr Trp Ala Asn Asp Thr Phe Gly 145 150 155 160 Ser Glu Lys Ala Ile Gln Leu Ile Ala Met Ala Thr Pro Glu Asp Met 165 170 175 Arg Ile Asn Ala Glu His Ile Arg Ile Ala Asp Gln Phe Val Glu Val 180 185 190 Pro Gly Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val Gln Leu Ile Val 195 200 205 Gly Met Ala Gln Lys Leu Gly Val Ser Ala Val Trp Pro Gly Trp Gly 210 215 220 His Ala Ser Glu Asn Pro Glu Leu Pro Asp Ala Leu Thr Ala Lys Gly 225 230 235 240 Ile Val Phe Leu Gly Pro Pro Ala Ser Ser Met Asn Ala Leu Gly Asp 245 250 255 Lys Val Gly Ser Ala Leu Ile Ala Gln Ala Ala Gly Val Pro Thr Leu 260 265 270 Ala Trp Ser Gly Ser His Val Glu Val Pro Leu Glu Cys Cys Leu Asp 275 280 285 Ala Ile Pro Glu Glu Met Tyr Arg Lys Ala Cys Val Thr Thr Thr Glu 290 295 300 Glu Ala Val Ala Ser Cys Gln Val Val Gly Tyr Pro Ala Met Ile Lys 305 310 315 320 Ala Ser Trp Gly Gly Gly Gly Lys Gly Ile Arg Lys Val His Asn Asp 325 330 335 Asp Glu Val Arg Ala Leu Phe Lys Gln Val Gln Gly Glu Val Pro Gly 340 345 350 Ser Pro Ile Phe Val Met Arg Leu Ala Ser Gln Ser Arg His Leu Glu 355 360 365 Val Gln Leu Leu Cys Asp Gln Tyr Gly Asn Val Ala Ala Leu His Ser 370 375 380 Arg Asp Cys Ser Val Gln Arg Arg His Gln Lys Ile Ile Glu Glu Gly 385 390 395 400 Pro Val Thr Val Ala Pro Arg Glu Thr Val Lys Ala Leu Glu Gln Ala 405 410 415 Ala Arg Arg Leu Ala Lys Ala Val Gly Tyr Val Gly Ala Ala Thr Val 420 425 430 Glu Tyr Leu Tyr Ser Met Glu Thr Gly Asp Tyr Tyr Phe Leu Glu Leu 435 440 445 Asn Pro Arg Leu Gln Val Glu His Pro Val Thr Glu Trp Ile Ala Glu 450 455 460 Val Asn Leu Pro Ala Ala Gln Val Ala Val Gly Met Gly Ile Pro Leu 465 470 475 480 Trp Gln Ile Pro Glu Ile Arg Arg Phe Tyr Gly Met Asp Tyr Gly Gly 485 490 495 Gly Tyr Asp Ile Trp Arg Lys Thr Ala Ala Leu Ala Thr Pro Phe Asn 500 505 510 Phe Asp Glu Val Asp Ser Gln Trp Pro Lys Gly His Cys Val Ala Val 515 520 525 Arg Ile Thr Ser Glu Asp Pro Asp Asp Gly Phe Lys Pro Thr Gly Gly 530 535 540 Lys Val Lys Glu Ile Ser Phe Lys Ser Lys Pro Asn Val Trp Ala Tyr 545 550 555 560 Phe Ser Val Lys Ser Gly Gly Gly Ile His Glu Phe Ala Asp Ser Gln 565 570 575 Phe Gly His Ala Phe Ala Tyr Gly Leu Ser Arg Pro Ala Ala Ile Thr 580 585 590 Asn Met Ser Leu Ala Leu Lys Glu Ile Gln Ile Arg Gly Glu Ile His 595 600 605 Ser Asn Val Asp Tyr Thr Val Asp Leu Leu Asn Ala Ser Asp Phe Arg 610 615 620 Glu Asn Lys Ile His Thr Gly Trp Leu Asp Thr Arg Ile Ala Met Arg 625 630 635 640 Val Gln Ala Glu Arg Pro Pro Trp Tyr Ile Ser Val Val Gly Gly Ala 645 650 655 Leu Tyr Lys Thr Val Thr Thr Asn Ala Ala Thr Val Ser Glu Tyr Val 660 665 670 Ser Tyr Leu Thr Lys Gly His Ile Pro Pro Lys His Ile Ser Leu Val 675 680 685 Asn Ser Thr Val Asn Leu Asn Ile Glu Gly Ser Lys Tyr Thr Ile Glu 690 695 700 Thr Val Arg Thr Gly His Gly Ser Tyr Arg Leu Arg Met Asn Asp Ser 705 710 715 720 Thr Val Glu Ala Asn Val Gln Ser Leu Cys Asp Gly Gly Leu Leu Met 725 730 735 Gln Leu Asp Gly Asn Ser His Val Ile Tyr Ala Glu Glu Glu Ala Gly 740 745 750 Gly Thr Arg Leu Gln Ile Asp Gly Lys Thr Cys Leu Leu Gln Asn Asp 755 760 765 His Asp Pro Ser Lys Leu Leu Ala Glu Thr Pro Cys Lys Leu Leu Arg 770 775 780 Phe Leu Val Ala Asp Gly Ala His Val Asp Ala Asp Val Pro Tyr Ala 785 790 795 800 Glu Val Glu Val Met Lys Met Cys Met Pro Leu Leu Ser Pro Ala Ser 805 810 815 Gly Val Ile His Cys Met Met Ser Glu Gly Gln Ala Leu Gln Ala Gly 820 825 830 Asp Leu Ile Ala Arg Leu Asp Leu Asp Asp Pro Ser Ala Val Lys Arg 835 840 845 Ala Glu Pro Phe Asp Gly Ile Phe Pro Gln Met Glu Leu Pro Val Ala 850 855 860 Val Ser Ser Gln Val His Lys Arg Tyr Ala Ala Ser Leu Asn Ala Ala 865 870 875 880 Arg Met Val Leu Ala Gly Tyr Glu His Asn Ile Asn Glu Val Val Gln 885 890 895 Asp Leu Val Cys Cys Leu Asp Asn Pro Glu Leu Pro Phe Leu Gln Trp 900 905 910 Asp Glu Leu Met Ser Val Leu Ala Thr Arg Leu Pro Arg Asn Leu Lys 915 920 925 Ser Glu Leu Glu Asp Lys Tyr Lys Glu Tyr Lys Leu Asn Phe Tyr His 930 935 940 Gly Lys Asn Glu Asp Phe Pro Ser Lys Leu Leu Arg Asp Ile Ile Glu 945 950 955 960 Glu Asn Leu Ser Tyr Gly Ser Glu Lys Glu Lys Ala Thr Asn Glu Arg 965 970 975 Leu Val Glu Pro Leu Met Asn Leu Leu Lys Ser Tyr Glu Gly Gly Arg 980 985 990 Glu Ser His Ala His Phe Val Val Lys Ser Leu Phe Glu Glu Tyr Leu 995 1000 1005 Thr Val Glu Glu Leu Phe Ser Asp Gly Ile Gln Ser Asp Val Ile Glu 1010 1015 1020 Thr Leu Arg His Gln His Ser Lys Asp Leu Gln Lys Val Val Asp Ile 1025 1030 1035 1040 Val Leu Ser His Gln Gly Val Arg Asn Lys Ala Lys Leu Val Thr Ala 1045 1050 1055 Leu Met Glu Lys Leu Val Tyr Pro Asn Pro Gly Gly Tyr Arg Asp Leu 1060 1065 1070 Leu Val Arg Phe Ser Ser Leu Asn His Lys Arg Tyr Tyr Lys Leu Ala 1075 1080 1085 Leu Lys Ala Ser Glu Leu Leu Glu Gln Thr Lys Leu Ser Glu Leu Arg 1090 1095 1100 Ala Ser Val Ala Arg Ser Leu Ser Asp Leu Gly Met His Lys Gly Glu 1105 1110 1115 1120 Met Ser Ile Lys Asp Asn Met Glu Asp Leu Val Ser Ala Pro Leu Pro 1125 1130 1135 Val Glu Asp Ala Leu Ile Ser Leu Phe Asp Tyr Ser Asp Arg Thr Val 1140 1145 1150 Gln Gln Lys Val Ile Glu Thr Tyr Ile Ser Arg Leu Tyr Gln Pro His 1155 1160 1165 Leu Val Lys Asp Ser Ile Gln Met Lys Phe Lys Glu Ser Gly Ala Ile 1170 1175 1180 Thr Phe Trp Glu Phe Tyr Glu Gly His Val Asp Thr Arg Asn Gly His 1185 1190 1195 1200 Gly Ala Ile Ile Gly Gly Lys Arg Trp Gly Ala Met Val Val Leu Lys 1205 1210 1215 Ser Leu Glu Ser Ala Ser Thr Ala Ile Val Ala Ala Leu Lys Asp Ser 1220 1225 1230 Ala Gln Phe Asn Ser Ser Glu Gly Asn Met Met His Ile Ala Leu Leu 1235 1240 1245 Ser Ala Glu Asn Glu Ser Asn Ile Ser Gly Ile Ser Ser Asp Asp Gln 1250 1255 1260 Ala Gln His Lys Met Glu Lys Leu Ser Lys Ile Leu Lys Asp Thr Ser 1265 1270 1275 1280 Val Ala Ser Asp Leu Gln Ala Ala Gly Leu Lys Val Ile Ser Cys Ile 1285 1290 1295 Val Gln Arg Asp Glu Ala Arg Met Pro Met Arg His Thr Phe Leu Trp 1300 1305 1310 Leu Asp Asp Lys Ser Cys Tyr Glu Glu Glu Gln Ile Leu Arg His Val 1315 1320 1325 Glu Pro Pro Leu Ser Thr Leu Leu Glu Leu Asp Lys Leu Lys Val Lys 1330 1335 1340 Gly Tyr Asn Glu Met Lys Tyr Thr Pro Ser Arg Asp Arg Gln Trp His 1345 1350 1355 1360 Ile Tyr Thr Leu Arg Asn Thr Glu Asn Pro Lys Met Leu His Arg Val 1365 1370 1375 Phe Phe Arg Thr Ile Val Arg Gln Pro Asn Ala Gly Asn Lys Phe Arg 1380 1385 1390 Ser Ala Gln Ile Ser Asp Ala Glu Val Gly Cys Pro Glu Glu Ser Leu 1395 1400 1405 Ser Phe Thr Ser Asn Ser Ile Leu Arg Ser Leu Met Thr Ala Ile Glu 1410 1415 1420 Glu Leu Glu Leu His Ala Ile Arg Thr Gly His Ser His Met Tyr Leu 1425 1430 1435 1440 Cys Ile Leu Lys Glu Gln Lys Leu Leu Asp Leu Ile Pro Phe Ser Gly 1445 1450 1455 Ser Thr Ile Val Asp Val Gly Gln Asp Glu Ala Thr Ala Cys Ser Leu 1460 1465 1470 Leu Lys Ser Met Ala Leu Lys Ile His Glu Leu Val Gly Ala Arg Met 1475 1480 1485 His His Leu Ser Val Cys Gln Trp Glu Val Lys Leu Lys Leu Asp Cys 1490 1495 1500 Asp Gly Pro Ala Ser Gly Thr Trp Arg Val Val Thr Thr Asn Val Thr 1505 1510 1515 1520 Gly His Thr Cys Thr Ile Asp Ile Tyr Arg Glu Val Glu Glu Ile Glu 1525 1530 1535 Ser Gln Lys Leu Val Tyr His Ser Ala Ser Ser Ser Ala Gly Pro Leu 1540 1545 1550 His Gly Val Ala Leu Asn Asn Pro Tyr Gln Pro Leu Ser Val Ile Asp 1555 1560 1565 Leu Lys Arg Cys Ser Ala Arg Asn Asn Arg Thr Thr Tyr Cys Tyr Asp 1570 1575 1580 Phe Pro Leu Ala Phe Glu Thr Ala Leu Gln Lys Ser Trp Gln Ser Asn 1585 1590 1595 1600 Gly Ser Thr Val Ser Glu Gly Asn Glu Asn Ser Lys Ser Tyr Val Lys 1605 1610 1615 Ala Thr Glu Leu Val Phe Ala Glu Lys His Gly Ser Trp Gly Thr Pro 1620 1625 1630 Ile Ile Pro Met Glu Arg Pro Ala Gly Leu Asn Asp Ile Gly Met Val 1635 1640 1645 Ala Trp Ile Met Glu Met Ser Thr Pro Glu Phe Pro Asn Gly Arg Gln 1650 1655 1660 Ile Ile Val Val Ala Asn Asp Ile Thr Phe Arg Ala Gly Ser Phe Gly 1665 1670 1675 1680 Pro Arg Glu Asp Ala Phe Phe Glu Thr Val Thr Asn Leu Ala Cys Glu 1685 1690 1695 Arg Lys Leu Pro Leu Ile Tyr Leu Ala Ala Asn Ser Gly Ala Arg Ile 1700 1705 1710 Gly Ile Ala Asp Glu Val Lys Ser Cys Phe Arg Val Gly Trp Ser Asp 1715 1720 1725 Glu Gly Ser Pro Glu Arg Gly Phe Gln Tyr Ile Tyr Leu Thr Glu Glu 1730 1735 1740 Asp Tyr Ala Arg Ile Ser Ser Ser Val Ile Ala His Lys Leu Glu Leu 1745 1750 1755 1760 Asp Ser Gly Glu Ile Arg Trp Ile Ile Asp Ser Val Val Gly Lys Glu 1765 1770 1775 Asp Gly Leu Gly Val Glu Asn Ile His Gly Ser Ala Ala Ile Ala Ser 1780 1785 1790 Ala Tyr Ser Arg Ala Tyr Glu Glu Thr Phe Thr Leu Thr Phe Val Thr 1795 1800 1805 Gly Arg Thr Val Gly Ile Gly Ala Tyr Leu Ala Arg Leu Gly Ile Arg 1810 1815 1820 Cys Ile Gln Arg Leu Asp Gln Pro Ile Ile Leu Thr Gly Phe Ser Ala 1825 1830 1835 1840 Leu Asn Lys Leu Leu Gly Arg Glu Val Tyr Ser Ser His Met Gln Leu 1845 1850 1855 Gly Gly Pro Lys Ile Met Ala Thr Asn Gly Val Val His Leu Thr Val 1860 1865 1870 Pro Asp Val Leu Glu Gly Val Ser Asn Ile Leu Arg Trp Leu Ser Tyr 1875 1880 1885 Val Pro Ala Asn Ile Gly Gly Pro Leu Pro Ile Thr Lys Pro Leu Asp 1890 1895 1900 Pro Pro Asp Arg Pro Val Ala Tyr Ile Pro Glu Asn Thr Cys Asp Pro 1905 1910 1915 1920 Arg Ala Ala Ile Cys Gly Val Asp Asp Ser Gln Gly Lys Trp Leu Gly 1925 1930 1935 Gly Met Phe Asp Lys Asp Ser Phe Val Glu Thr Phe Glu Gly Trp Ala 1940 1945 1950 Lys Thr Val Val Thr Gly Arg Ala Lys Leu Gly Gly Ile Pro Val Gly 1955 1960 1965 Val Ile Ala Val Glu Thr Gln Thr Met Met Gln Ile Ile Pro Ala Asp 1970 1975 1980 Pro Gly Gln Leu Asp Ser His Glu Arg Ser Val Pro Arg Ala Gly Gln 1985 1990 1995 2000 Val Trp Phe Pro Asp Ser Ala Thr Lys Thr Ala Gln Ala Leu Leu Asp 2005 2010 2015 Phe Asn Arg Glu Gly Leu Pro Leu Phe Ile Leu Ala Asn Trp Arg Gly 2020 2025 2030 Phe Ser Gly Gly Gln Arg Asp Leu Phe Glu Gly Ile Leu Gln Ala Gly 2035 2040 2045 Ser Thr Ile Val Glu Asn Leu Arg Thr Tyr Asn Gln Pro Ala Phe Val 2050 2055 2060 Tyr Ile Pro Met Ala Gly Glu Leu Arg Gly Gly Ala Trp Val Val Val 2065 2070 2075 2080 Asp Ser Lys Ile Asn Pro Asp Arg Ile Glu Cys Tyr Ala Glu Arg Thr 2085 2090 2095 Ala Lys Gly Asn Val Leu Glu Pro Gln Gly Leu Ile Glu Ile Lys Phe 2100 2105 2110 Arg Ser Glu Glu Leu Gln Asp Cys Met Gly Arg Leu Asp Pro Glu Leu 2115 2120 2125 Ile Asn Leu Lys Ala Lys Leu Gln Asp Val Asn His Gly Asn Gly Ser 2130 2135 2140 Leu Pro Asp Ile Glu Gly Ile Arg Lys Ser Ile Glu Ala Arg Thr Lys 2145 2150 2155 2160 Gln Leu Leu Pro Leu Tyr Thr Gln Ile Ala Ile Arg Phe Ala Glu Leu 2165 2170 2175 His Asp Thr Ser Leu Arg Met Ala Ala Lys Gly Val Ile Lys Lys Val 2180 2185 2190 Val Asp Trp Glu Glu Ser Arg Ser Phe Phe Tyr Lys Arg Leu Arg Arg 2195 2200 2205 Arg Ile Ala Glu Asp Val Leu Ala Lys Glu Ile Arg Gln Ile Val Gly 2210 2215 2220 Asp Lys Phe Thr His Gln Leu Ala Met Glu Leu Ile Lys Glu Trp Tyr 2225 2230 2235 2240 Leu Ala Ser Gln Ala Thr Thr Gly Ser Thr Gly Trp Asp Asp Asp Asp 2245 2250 2255 Ala Phe Val Ala Trp Lys Asp Ser Pro Glu Asn Tyr Lys Gly His Ile 2260 2265 2270 Gln Lys Leu Arg Ala Gln Lys Val Ser His Ser Leu Ser Asp Leu Ala 2275 2280 2285 Asp Ser Ser Ser Asp Leu Gln Ala Phe Ser Gln Gly Leu Ser Thr Leu 2290 2295 2300 Leu Asp Lys Met Asp Pro Ser Gln Arg Ala Lys Phe Val Gln Glu Val 2305 2310 2315 2320 Lys Lys Val Leu Asp 2325 22 base pairs nucleic acid single linear cDNA NO NO unknown 7 GGTCTTCAAT TGTGCTGTCT GG 22 24 base pairs nucleic acid single linear cDNA NO NO unknown 8 CCTTGACGAA CAGACTGGCT GTGC 24 23 base pairs nucleic acid single linear cDNA NO NO unknown 9 CACAGCCAGT CTGTTCGTCA AGG 23 20 base pairs nucleic acid single linear cDNA NO NO unknown 10 CCTCTACGTA ATTGGTCAGC 20 20 base pairs nucleic acid single linear cDNA NO NO unknown 11 CATAGCTATG GCAACTCCGG 20 3488 base pairs nucleic acid single linear Genomic DNA NO NO unknown 12 AAGCTTGGTA TGGATTCGTC AGCGCCAAGC CGGGGTTTTG CATGCGCCCG ACTGGAARCS 60 GAATTCCGTG AGCCCTGTAC RRCAATGGCA ACCCCASGGT TACTGGGGTG GCTGAATGGT 120 CTCSGCTTAC GCAATTGTTT GTGGCAGCWG CGTGGGCTAA ATGTARGTTG TCTCTTGTTG 180 CACTGCARGA TGGATGGGTA GCCTCTGGGC CGCCTCTGCT ARTGTCTARC GTTTGCTGAC 240 TGTGGTTTAT TCAGGGATGC CCATGCCCAT GCTAGATTGA TAGGTGCCAT TCTAATGGTA 300 GGTGGCGGTA AGGTTTATTA AGCTGYAGYA TCAGTAGGTA ACCTCATGAA TCAGGGTTTA 360 AGCACACCTT TTCCTTTGTG TGGGTGCATA AGGAATGCAC TTGGCTTCGT TCCCTGATAG 420 TCTTTGSTCA TGTGTCATTC TACCAAGTGG GTTACTGTAA CATTGCACTC TATGATGGTT 480 GGTGGTTGTG CATCTTTYTG CTYCCCCTGG YTGTCTAATA CCTGCATGTA ACTGATGACC 540 YYCYTTTATG TATCATATAG ATTACATCCT TTTGTTGTAC ATCTCAATTC TGAAAAAACA 600 ATGTTTTGCA TTCTTAGCGC TCTGTGCACA AGGAAAAGGA GGTTTTACCT GCAACTTTTT 660 TTTTCGAGAA AAAACAAACC TTTCTGAAAG GCAGTGATCA TTTAGTATAA AGAAAATTTG 720 ATTTACTTTC TTCAGAGAGA ATATKCCAAR CAAACAATTT TCTTACTGTC TGAGCCACGA 780 AATTTGATCT TGATCTTACT TTCACAAGCC ACATGAAGCC TTATCATCGC TCTGATAAAA 840 AARCCAAATA GGTGATTCAT AGAATGAGAR AAAGAACCTG TTGCCATTTG GGGACCTTGT 900 TGTGTACTCA TTATCCCCCC TGCTCAGGTT GAGGTTTCCT TGCCACTGCC ACCCCTTGGC 960 CCCTTCTTAT ACAACCATCT CCATTGAAAA AGATTTTGCA CTACATTTGG GCTTCGTATG 1020 ACAAAAAAGG AAAATAAAAC TAAACAGCAG AAACATAGTA TAATTATAGG TAAAAGGTTC 1080 TGGCAAGTTT GAGTGGAAGA GACCTTTGTA TATTTGGACA TATTTCACTA GTAAATAGTT 1140 TTCTAAAATC TTCATGAATG GTGGCCAATA AACTTGATAA GATCTCAACA TGGCAGGTTC 1200 CTTCMAAATG AGAGGAAAAC TGGAAACATC ACAAATATTT TTTAGCGAGT GGCCTATAAA 1260 TTATAATGTT GCTTTCATTT CTTTGATATT CAAAACTTCC TAAGAGTATT CTGCTAGAGC 1320 TCTGATGGTG TCTTTTGCCT CTGTCAGATT TTCCAGGAGT TTTCTTCCCT TTTTATGGCA 1380 CTGTGCGTTT GAGAAGGTCT TCAATTGTGC TGTCTGGGCC ACGGAACGAC AATGTCACAG 1440 CTTGGATTAG CCGCAGCTGC CTCAAAGGCC TTGCCACTAC TCCCTAATCG CCAGAGAAGT 1500 TCAGCTGGGA CTACATTCTC ATCATCTTCA TTATCGAGGC CCTTAAACAG AAGGAAAAGC 1560 CATACTCGTT CACTCCGTGA TGGCGGAGAT GGGGTATCAG ATGCCAAAAA GCACAGCCAG 1620 TCTGTTCGTC AAGGTACTGT GAATATCTTT TGATACAAGC TAAAATTTTG CTACAGAATA 1680 TATATTTAAA GAGTTCTTTC TTGGCTGGTG TTGTTTATTT GTTTAACATS CGAAAGGGCC 1740 TCTAGTTGGA TTGGTTAGGT GGSCTGAATA CCACTCCTTA AGGTCTTGAG TTTGCTTTTC 1800 CCCNCGGAGC GAATTTTAGG CTAGGGTTAC CCCCCCACCC CCACCCGAAT CTGCACAGYC 1860 CGGYCGYGGY CGYCCTCATA TAGGCTACGA TGTCATTGTG TATCGGCGGG CCAGGGGTTT 1920 AAGAGTTTTC TTGACCTTTG TTAGAAGATC TTAATAATAC AATGTCCAAG GGCTGTCTTA 1980 CCCTGTAGGT CGAGTTTTTA GTTGTTTTAA CATGGTAATG TTTGAAGCCT CATTCTAGGT 2040 RCCAATATAG ATATGCTCAC TGCTCAGTTT CAAATGTTTG TCTGCATGTA GGTCTTGCTG 2100 GCATTATCGA CCTCCCAAGT GAGGCACCTT CCGAAGTGGA TATTTCACAG TAAGGACTAC 2160 AATATTTTGC GTACGTTTGT TTTGGAAAAA GAAAATATTC TCAGCTTATT TATACTAGCT 2220 TCGCTAATAC TGAAATGCTG TCTTAATGTC CTGGTGCTGT ATGCTCAATC TTTCATAGTA 2280 AATGCTGCAA AATATGTGAT GTAACTGTTG CAACACAGCC AGGGACCTGT TATTTAGAGC 2340 ATGGTGAATG CTCTGGTTCA GTTATATGAT GTAGTTATAG CTCATGTTGA AGAATTAGTT 2400 GCAGTGTTTG CTGGACAATG GTCACTTATT ATAAATCATA TCTGCATACA CATTTGTGAC 2460 TTCTGTTGCT GTAAATGCCC GCATTTTTTG AGAAAAATTT AAATGCTTGG CCTAAATTGG 2520 ACATATATGA TAGACAAAGC TGATTTGAAC TTTGTTTATT TTTGACATCC ATGCATATTG 2580 TCAGTGTTGT GAAAACAATA CTAATCCTTT TTTTTTGTCT TTTTCCAGTG GATCTGAGGA 2640 TCCTAGGGGG CCAACAGATT CTTATCAAAT GAATGGGATT ATCAATGAAA CACATAATGG 2700 AAGACATGCC TCAGTGTCCA AGGTTGTTGA ATTTTGTGCG GCACTAGGTG GCAAAACACC 2760 AATTCACAGT ATATTAGTGG CCAACAATGG AATGGCAGCA CCCAAATTTA TGAGGAGTGT 2820 CCGGACATGG GCTAATGATA CTTTTGGATC TGAGAAGGCA ATTCAACTCA TAGCTATGGC 2880 AACTCCGGAA GACATGAGGA TAAATGCAGA ACACATTAGA ATTGCTGACC AATTACGTAG 2940 AGGTGCCTGG TGGAACAAAC AATAATAACT ACGCCAATGT TCAACTCATA GTGGAGGTTA 3000 GCCTTGCTAA TCTGTTAGTT TACTACTGGT CTGCTGTTTC CTTTATTTGT TGTATAATGA 3060 TTGACATATT TAAGTAGAGA AATTTATATT TCTCCTCTGC TGTTGTGGAA GTCCAATTGT 3120 CATCATTAAC TGTGAAATAT TGCAGATGGC ACAAAAACTA GGTGTTTCTG CTGTTTGGCC 3180 TGGTTGGGGT CATGCTTCTG AGAATCCTGA ACTGCCAGAT GCATTGACCG CAAAAGGGAT 3240 CGTTTTTCTT GGCCCACCTG SATCATCAAT GAATGCTTTG GGAGATAAGG TCGGCTCAGC 3300 TCTCATTGCT CAAGCAGCCG GGGNCCCAAC TCTTGCTTGG AGTGGATCAC ATGTGAGTCT 3360 CACTCTTTGA TTACTATCCG CCTGTCTCAT TGCTCTCTCT TTCATATTCT AATGACACTA 3420 AATTTAGGTT GAAGTTCCAT TAGAGTGCTG CTTAGACGCG ATACCTGAGG AGATGTATAG 3480 AAAAGCTT 3488 1328 base pairs nucleic acid single linear Genomic DNA NO NO unknown 13 GAATTCCGTG AGCCCTGTAC GGCAATGGCA ACCCCAGGGT TACTGGGGTG GCTGAATGGT 60 CTCGGCTTAC GCAATTGTTT GTGGCAGCTG CGTGGGCTAA ATGTAGGTTG TCTCTTGTTG 120 CACTGCAGGA TGGATGGGTA GCCTCTGGGC CGCCTCTGCT AGTGTCTAGC GTTGCTGACT 180 GTGGTTTATT CAGGGATGCC CATGCCCATG CTAGATTGAT AGGTCATAGG TGCCATTCTA 240 ATGGTAGGTG GCGGTAAGGT TTATTAAGCT GTCGTATCAG TAGGTAACCT CATGAATCAG 300 GGTTTAAGCC CACCTTCTCC TTTGTGTGGG TGCATAAGGA ATGCACTTGG CTTCGTTCCC 360 TGCTAGTCTT TGCTCATGTG TCATTCTACC AAGTGGGTTA CTGTAACATT GCACTCTATG 420 ATGGTTGGTG GTTGTGCATC TTTTTGCTTC CCCTGGTTGT CTAATACCTG CATGTAACTG 480 ATGACCTTCT TTTATGTATC ATATAGATTA CATCTTTTGT TGTACATCTC AATTCTGAAA 540 AACAATGTTT TGCATTCTTA GCGCTCTGTG CACAAGGAAA AGGAGGTTTT ACCTGCAACT 600 TTTTTTTTCG AGAAAAAACA AACCTTTCTG AAAGGCAGTG ATCATTTAGT ATAAAGAAAA 660 TTTGATTTAC TTTCTTCAGA GAGAATATTC CAAACAAACA ATTTTCTTAC AGTCTGAGCC 720 ACGAAATTTG ATCTTGATCT TACTTTCACA AGCCACATGA AGCCTTATCA TCGCTCTGAT 780 AAAAAAACCA AATAGGTGAT TCATAGAATG AGAAAAAGAA CCTGTTGCCA TTTGGGGACC 840 TTGTTGTGTA CTCATTATCC CCCCTGCTCA GGTTGAGGTT TCCTTGCCAC TGCCACCCCT 900 TGGCCCCTTC TTATACAACC ATCTCCATTG AAAAAGATTT TGCACTACAT TTGGGCTTCG 960 TATAACAAAA AAGGAAAATA AAACTAAACA GCAGAAACAT AGTATAATTA TAGGTAAAAG 1020 GTTNTGGCAA GTTTGAGTGG TAGAGACCTT TGTATATTTG GACATATTTC ACTAGTAAAT 1080 AGTTTTCTAA AATGTTCATG AATGGTGGCC AATAAACTTG ATAAGATCTC AACATGGCAG 1140 GTTCCTTCAA AATGAGAGGA AAACTGGAAA CATCACAAAT ATTTTTTAGC GAGTGGCCTA 1200 TAAATTATAA TGTTGCTTTC ATTTCTTTGA TATTCAAAAC TTCCTAAGAG TATTCTGCTA 1260 GAGCTCTGAT GGTGTCTTTT GCCTCTGTCA GATTTTCCAG GAGTTTTCTT CCCTTTTTAT 1320 GGCACTGT 1328 1565 base pairs nucleic acid single linear Genomic DNA NO NO unknown 14 AGCATCCCTT GGGATTGTGA TNACTCACAT AAATTCTTGC GAANTGTTGA CATTCTAGTG 60 ATTTGAGTTC CGTTCTAGTG TGCTAGTCAN TTGAGCTCAA GTCTTGGTTT TATGTGTGCG 120 TATTCACTGT GATCTTTGTG TCGTGTGTGA GTTGTTGATC CTTCCCTTGC TCCGTGATTC 180 TTTGTGAAAT CTTTTGAAAG GGCGAGAGGC TCCAAGCTGT GGAGATTCCT CGCAAGTGGG 240 ATTAAGAAAA GCAAAGCAAC ACCGTGGTAT TCAAGTTGGT CTTTGGACCG CTTGAGAGGG 300 GTTGATTGCA ACCCTCGTCC GTTGGGACGC CACAACGTGG AGTAGGCAAG CGTTGGTCTT 360 GGCCGAACCA CGGGATAACC ACCGTGCCAT CTCTGTGATT GATATCTCTT GGTTATTGTG 420 TTGTGTTGAG ATCCTTCTCT AGCCACTTGG CAAATTACTG TGCTAACAAT TAATCAAGTT 480 TTGTGGCTTA AGATTTTGAA GTATTACAGG ATCTGCATCA TGGTCTGTGT CTCCACAGCT 540 ATGACACCCA CAGGAATTCA TGTGTTCCTT GGAGCCACTC TTGGATGACC TAAAGGAATT 600 ATTTCTAACC GGCTTGTACA CATATGATGC ATCAAGAGAT GAGTGTTTTA CTATGCGAGG 660 GGCCATGCTT ATGACCATAA GTAATCTTCC TGGTTTAGAA ATGCTTGCTT CTCATATGGT 720 TCATGGGAAA TTCGCATGCC TCCTTGTGGT GAAAATGTCT GGACAAAACA GCTGAAGAAT 780 GGTCGTAAAT CTTGTTTTAT GGGAAATCGC CAATATATTG ATCTTGATCA TTCTTATTGC 840 TTGGATGCAG ACTCCGTTTG ATGGAACGAT AGACTTCGAA CAAAACCTAA AACCTATTAT 900 GATCGTCCAA TTTTGGATGA AATCATCACA CTTGGTGATT TCAAGAACTC AAAAAYTTAC 960 AGTTAATTGG ATATAGGAGG GNGCAAAAAC ACAGTAAGTT GGACATTCCA TAAGGGGATT 1020 TATTTTAGTT GACAATAAAG TAGATGGGCA TCATCCTGAG TTTNGTTTGG CATCGTGTCG 1080 TAGATTGAAA CTGTAAGGAT GGACATGGTA GNTAACAGGT TGAGATGAAT GATTCAACAG 1140 TTGAAGCGAA TGTACAATCT TTATGTGATG GTGGCTNTTA ATGCAGGTAA CTAGTTTTTT 1200 TTTATGCTTT ATTATTAATT AGTTGGATAA ATGGTTTNGA TTTNTGATTG TTAAANTGCA 1260 ATGGCTCCAG TTGGATGGNA ACAGCCANGT AATTTATGCA GAAGNAGNAG NTGGTGGTAC 1320 ACGGNTTCAG ATTGATGGAA AGANATGTTT ATTGCAGGTA AATANTCCCT TNTTCCTTTA 1380 TATTTTTGTT GTNTGATTGT ATAANTTTGN TAGATTATTT GTATAATTTA TTATTGCATT 1440 TCACCCCACT AANTTATTTT TAAAAGATGG GTTTTGTTGT TTGNTTCAGC NGGCGACATC 1500 ACATAAGNAA ATTGTGATTA ATTTTTGTTT TTTTGCAGNA TGACCATGAT CCATCAAAGT 1560 TATTA 1565 1168 base pairs nucleic acid single linear Genomic DNA NO NO unknown 15 ACATAAGCTG GGTTAGTAGT GGTGAATTAG TGGATTATTG ATCTGTTGTG GGCAAGGAGN 60 TGGCTTGGTG TCGAGAAAAA ACATGGAAGT GTGTATTGCC AGTGCTTATT YTRGGGATAT 120 GAGGGAATTW AMATTACATT TGTGACTGGG CGGACTGTAG GATAGGAGTT ATCTTGYTCG 180 ATTGGTATAC GGTGCATACA GSKYTTGACC AGCTATTATT TTAACAGGGT TTTCTGCCCT 240 GAACAAGTCC TTGGGCGGGA AGTGTACAGC TCCCACATGC AGCTTGGTGG TCCTAAGATC 300 ATGGCGACCA ATGGTGTTGT CCACCTCACT GTTCCAGATG ACCTTGAAGG TGTTTCCAAT 360 ATATTGAGGT GGCTCAGCTA TGTTCCTGCA AACATTGGTG GACCTCTTCC TATTACCAAA 420 CCTCTGGACC CTCCAGACAG ACCTGTTGCT TACATCCCTG AGAACACATG CGATCCACGT 480 GCAGCTATCT GTGGTGTAGA TGACAGCCAA GGGAAATGGT TGGGTGGTAT GTTTGACAAA 540 GACAGCTTTG TGGAGACATT TGAAGGATGG GCAAAAACAG TGGTTACTGG CAGAGCAAAG 600 CTTGGAGGAA TTCCATGCAT CTTAATAAAC ACAGTTGGCC CTTAAAGCAA GTGAACTTCT 660 TGAACAAACC AAACTAAGTG AACTCCGTGC AAGCGTTGCA AGAAGCCTTT CGGATCTGGG 720 GATGCATAAG GGAGAAATGA GTATTAAGGA TAACATGGAA GATTTAGTCT CTGCCCCATT 780 ACCTGTTGAA GATGCTCTGA TTTCTTTGTT TGATTACAGT GATCGAACTG TTCAGCAGAA 840 AGTGATTGAG ACATACATAT CACGATTGTA CCAGGTATTA TATCAACTAA CTTAATGTCT 900 TCCATAGTCT CACTAAGCAT ATCTGATATG TTTAGATACC CTACATGGAA TGCTCATCTT 960 TTCATTTGAC ACAAAGAAAC ATTGAGAAAT GAGATGCTGA CGATTGGCTG AAATTAACTG 1020 GGTNTGAGAA ATTGTGATCT CCCAACTTGT TAATGCACAA TGTTCTGGCT AACTTGCCAA 1080 TATTTTTTCA GCCTCATCTT GTTNAGGATA GCANCCAAAT GAAATCCAAG GATCTGGTGC 1140 TATTACTTTT TGGGAATTTA TGAAGGGC 1168 638 base pairs nucleic acid single linear Genomic DNA NO NO unknown 16 CTCCCAATAT TGTCATGAGG CTTGCATCCC AGGTTAGTTT TTTTTCCTTT CTGAAATTTA 60 TATTCCATAC CTTTTCACCT TTAGTTATCC TTGTATTTTC TGGAAGCTTC ATCTGATGCA 120 TTATTGACAA ATGCACTAAT GGTCATCATA TTTGGAGATT AACATATTTA TCTTAATTGA 180 TGGGAACTCT TGAAAATGAC AATGGTTGAG CAGATAATTA ACAGTTTTTT AATAAAAAAA 240 CATGCATTTC TAGGAGTTGG ACTAAGCTTT TCTTAGTATG AAGTGCCATG TTTTACATGG 300 TCCATTTGTG TCAATTTACA GTCGGTATCA TGGAAAGGTT GTCATAATGG CTGGAGANAA 360 ACAACACATC TTGTTTCTCA ACACTTGTGG GAGAAGANGT TTTACCTTTT TTCCTAAAAT 420 TACTTTTTGT ACTAAATTGT ATAATTTTTC CAATATTCTC CATGATTATT GAACTCTGCT 480 GTGTTCAAAC AGCCAAAACA TGTTTCCATA CTTTACACCT TTATTTTTTA GATGGAACCT 540 GGAATTGTGC TCTGTTATCT GTATCATGCA TATATTGATC TTAAACCTAT CTCTATTGTA 600 GAATCCGCAC TTGAATTCAG TTGCTTGTGA TCAATATG 638 558 base pairs nucleic acid single linear Genomic DNA NO NO unknown 17 GGTAACCACC ACACCCGCGG CGCTTAATGG CCGTACAGGG NGGTCCCATT CGCCATTCAG 60 GTGCGCAACT GTTGGGAAGG GCGATCGGTG CGGGCTTCTT CGNTATTACG CCAGCTGGCG 120 AAAGGGGGAT GTGCTGCAAG GCGATTAAGT TGGGTAACGC CAGGGTTTTC CCAGTCACGA 180 CGTTGTAAAA CGACGGCCAG TGAGCGCGCG TAATACGACT CACTATAGGG CGAATTGGGT 240 ACCGGGCCCC CCCTCGAGGT CGACCTGCAG GTCAACGGAT CCTAGGGGGC CAACAGATTC 300 TTATCAAATG AATGGGATTA TCAATGAAAC ACATAATGGA AGACATGCCT CAGTGTCCAA 360 GGTTGTTGAA TTTTGTGCGG CACTAGGTGG CAAAACACCA ATTCACAGTA TATTAGTGGC 420 CAACAATGGA ATGGCAGCAG CAAAATTTAT GAGGAGTGTC CGGACATGGG CTAATGATAC 480 TTTTGGATCT GAGAAGGCAA TTCAACTCAT AGCTATGGCA ACTCCGGAAG ACATGAGGTA 540 AATGCAGAAC ACATTAGA 558 975 base pairs nucleic acid single linear Genomic DNA NO NO unknown 18 GAATAATCTG CCTGCAGCTC AAGTTGCTGT TGGAATGGGC ATACCTCTTT GGCAGATTCC 60 AGGTAATTAC CAATTTACCA ACTTATTTAG TTCCTTATTG TTTTATTCTC TAATTTTCTA 120 CTTATGTAGA AATCAGACGT TTCTATGGAA TGGACTATGG AGGAGGGTAT GACATTTGGA 180 GGAAAACAGC AGCTCTTGCT ACACCATTTA ATTTTGATGA AGTAGATTCT CAATGGCCAA 240 AGGGCCATTG TGTAGCAGTT AGAATTACTA GTGAGGACCC AGATGATGGT TTCAAACCTA 300 CTGGTGGGAA AGTGAAGGTA AGTTTTCTAG ATGACATGTA TTATATATCG TTCAAAGAGA 360 TTAAGTTTGG TTAAATGACT AGGTCTTGAT TTTTTATCTT TCAGGAGATA AGTTTTAAAA 420 GCAAGCCTAA TGTTTGGGCC TACTTCTCAG TAAAGGTAAC TTGTTAACTT TAGTACGCTG 480 TCACATTATT CTTCSTTGTG AAAATAATTT GAACGGTTCT CTTTGTATTT TAACCATCCA 540 TCGTCTCATT TASCAGAGCA CACAAATATT TGCACTGACC CCCCTCCCCT TATCTGCTTT 600 CAGTCTGGTG GAGGCATTCA TGAATTTGCT GATTCTCAGT TCGGTATGTG TAAACCAAGA 660 GTATTCTTTG TAATTTATAT TGGTCCTCAA TTTTGAAATA TTGCTCTTTC CGTTACAGGA 720 CAWGTTTTTG CATATGGGCT CTCTAGATCA GCAGCAATAA CAAACATGAC TCTTGCATTA 780 AAANAGATTC AAATTCGTGG AGAAATTCAT TCAAATGTTT GATTACACAG TTGATCTCTT 840 AAATGTTAAG AAATATTAAC CACCTTTTAA ATCACATTTT CCATTATGTT TGATTCCATA 900 TCATTAATTT TGATTTTCTA TTATGGCTAA ACCTGTGGTG CTATTTTCCT ATTATCCCAG 960 GCTTCCGACT TTAGA 975 852 base pairs nucleic acid single linear Genomic DNA NO NO unknown 19 GGATCCTAGG GGGCCAACAG ATTCTTATCA AATGRAATGG GATTATCAAT GAAACACATA 60 ATGGAAGACA TGCCTCAGTG TCCAAGGTTG TTGAATTTTG TGCGGCACTA GGTGGCAAAA 120 CACCAATTCA CAGTATATTA GTGGCCAACA ATGGAATGGC AGCAGCAAAA TTTATGAGGA 180 GTGTCCGGAC ATGGGCTAAT GATACTTTTG GATCTGAGAA GGCAATTCAA CTCATAGCTA 240 TGGCAACTCC GGAAGACATG AGGATAAATG CAGAACACAT TAGAATTGCT GACCAATTAC 300 GTAGARGTGC CTGGTGGAAC AAACAATAAT AACTACGCCA ATGTTCAACT CATAGTGGAA 360 GTTAGCCTTG CTAATCTGTT AGTTTACTAC TGGTCTGCTG TTTCCTTTAT TTGTTGTATA 420 ATGATTGACA TATTTAAGTA GAGAAATTTA TATTTCTCCT CTGCTGTTGT GGAAGTCCAA 480 TTGTCACCAT TAACTGTGAA ATATTGCAGA TGGCACAAAA ACTAGGTGTT TCTGCTGTTT 540 GGCCTGGTTG GGGTCATGCT TCTGAGAATC CTGAACTGCC AGATGCATTG ACCGCAAAAG 600 GGATCGTTTT TCTTGGCCCA CCTGCATCAT CAATGAATGC TTTGGGAGAT AAGGTCGGCT 660 CAGCTCTCAT TGCTCAAGCA GCCGGGGTCC CAACTCTTGC TTGGAGTGGA TCACATGTGA 720 GTCTCACTCT TTGATTACTA TCCGCCTGTC TCATTGCTCT CTCTTTCATA TTCTAATGAC 780 ACTAAATTTA GGTTGAAGTT CCATTAGAGT GCTGCTTAGA CGCGATACCT GAGGAGATGT 840 ATAGAAAAGC TT 852 231 base pairs nucleic acid single linear Genomic DNA NO NO unknown 20 AATTCCTGTG GGTGTTATAG CTGTGGAGAC ACAGACCATG ATGCAGCTCA TCCCTGCTGA 60 TCCAGGTCAA CTTGATTCCC ATGAGCGATG TGTTCCTCGG GCTGGACAAG TGTGGTTCCC 120 AGATNCTGCA ACCAAGACAG CTCAGGCATT ATTAGACTTC AACCGTGAAG GATTGCCTCT 180 GTTCATCCTG GCTAACTGGA GAGGCTTCTC TGGGGGACAG AGAGATCTCT T 231 207 base pairs nucleic acid single linear Genomic DNA NO NO unknown 21 AATTCATGCA TCTTAATAAA CACAGTTGGC CCTTAAAGCA AGTGAACTTC TTGAACAAAC 60 CAAACTAAGT GAACTCTGTT CCAGCATTGC AAGAAGCCTT TCAGATCTGG GGATGCATAA 120 GGGAGAAATG ACTATTAAGG ATAGCATGGA AGATTTAGTC TCTGNCCCAT TGCCTGTTGA 180 AGATGCTCTT ATTTCTTTGT TTGATTA 207 180 base pairs nucleic acid single linear Genomic DNA NO NO unknown 22 ATAGACCTGT CGCATACATC CCTGAGAACA CATGCGATCC GCGTGCAGCC ATCCGTGGNG 60 TAGATGACAG CCAAGGGAAA TGGTTGGGTG GTATGTTTGA CAAAGACAGC TTTGTGGAGA 120 CATTTGAAGG ATGGGCAAAA ACAGTGGTTA CTGGTAGAGC AAAGCTTGGA GGAAGGAATT 180 20 base pairs nucleic acid single linear cDNA NO NO unknown 23 ACTGTGCGTT TGAGAAGGTC 20 20 base pairs nucleic acid single linear cDNA NO NO unknown 24 CCTCTACGTA ATTGGTCAGC 20 20 base pairs nucleic acid single linear cDNA NO NO unknown 25 CCTTTTTATG GCACTGTGCG 20 22 base pairs nucleic acid single linear cDNA NO NO unknown 26 CATCGTAGCC TATATGAGGA CG 22 

What is claimed is:
 1. A method for altering the oil content of plant cells comprising: (a) introducing an expression cassette comprising a promoter functional in a plant cell operably linked to a DNA molecule encoding a maize acetyl CoA carboxylase comprising an amino terminal chloroplast transit peptide, into the cells of a plant so as to yield transformed plant cells; and (b) regenerating said transformed plant cells to provide a differentiated transformed plant, wherein expression of the DNA molecule encoding the maize acetyl CoA carboxylase in said plant alters the oil content of the plant cells relative to the oil content in cells of a corresponding untransformed plant.
 2. The method according to claim 1, wherein the acetyl CoA carboxylase is expressed in an amount that is greater than that in the untransformed plant.
 3. The method according to claim 1, wherein the oil content of the transformed plant cells is increased.
 4. The method according to claim 1, wherein the DNA molecule comprises SEQ ID NO:5.
 5. The method according to claim 1, wherein the acetyl CoA carboxylase comprises SEQ ID NO:6.
 6. The method according to claim 1, wherein the DNA molecule encoding the acetyl CoA carboxylase encodes a variant acetyl CoA carboxylase, wherein the variant acetyl CoA carboxylase has a specific activity which is different from the specific activity of the native acetyl CoA carboxylase.
 7. The method of claim 1 wherein the DNA molecule comprises SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22.
 8. A method for altering the oil content of plant cells comprising: (a) introducing into plant cells an expression cassette comprising a promoter functional in a plant cell operably linked to a DNA molecule which is complementary to a DNA comprising SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22 so as to yield transformed plant cells; and (b) regenerating said transformed plant cells to provide a differentiated transformed plant, wherein expression of the DNA molecule in said plant alters the oil content of the plant cells relative to the oil content in the cells of a corresponding untransformed plant.
 9. The method according to claim 1 or 8, wherein the promoter is a maize globulin promoter.
 10. The method according to claim 1 or 8, wherein the promoter is a maize ubiquitin promoter.
 11. A transformed plant prepared by the method of claim
 1. 12. A transformed seed of the transformed plant of claim
 11. 13. A transformed plant having an altered oil content in its cells comprising a recombinant DNA molecule comprising a promoter functional in a plant cell operably linked to a DNA encoding a maize acetyl CoA carboxylase comprising an amino terminal chloroplast transit peptide, wherein expression of the DNA molecule in the cells of the plant alters the oil content of the plant cells relative to the oil content in the cells of a corresponding untransformed plant.
 14. The transformed plant of claim 13, wherein the transformed plant has an increase in oil content in its leaves, seeds, or fruit above that present in a corresponding untransformed plant.
 15. The transformed plant of claim 13, wherein the DNA molecule comprises SEQ ID NO:5.
 16. The transformed plant of claim 13, wherein the acetyl CoA carboxylase comprises SEQ ID NO:6.
 17. The transformed plant of claim 13, wherein the DNA molecule encodes a variant acetyl CoA carboxylase, wherein the variant acetyl CoA carboxylase has a specific activity which is different from the specific activity of the native acetyl CoA carboxylase.
 18. The transformed plant of claim 13, which is a dicot.
 19. The transformed plant of claim 13, which is a monocot.
 20. The plant of claim 13 wherein the DNA molecule comprises SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22.
 21. A transformed seed of the transformed plant of claim
 13. 22. A transformed plant having an altered oil content in its cells comprising: a recombinant DNA molecule comprising a promoter functional in a plant cell operably linked to a DNA which is complementary to a DNA comprising SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22 wherein expression of the DNA molecule in the cells of the plant alters the oil content of the plant cells relative to the oil content in the cells of a corresponding untransformed plant.
 23. The transformed plant of claim 13 or 22, wherein the promoter is a maize globulin promoter.
 24. The transformed plant of claim 13 or 22, wherein the promoter is a maize ubiquitin promoter.
 25. A transformed plant prepared by the method of claim
 8. 26. A transformed seed of the transformed plant of claim
 25. 27. The transformed seed of claim 12 or 26, wherein the transformed seed has an altered oil content relative to the oil content of an untransformed seed.
 28. The transformed plant of claim 13 or 22, wherein the transformed plant has an altered oil content in its seeds relative to the oil content of an untransformed plant.
 29. An expression cassette comprising a maize globulin promoter operably linked to a DNA molecule encoding a maize acetyl CoA carboxylase comprising an amino terminal chloroplast transit peptide.
 30. An expression cassette comprising a maize ubiquitin promoter operably linked to a DNA molecule encoding a maize acetyl CoA carboxylase comprising an amino terminal chloroplast transit peptide.
 31. The expression cassette of claim 29 or 30 wherein the DNA molecule comprises SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22.
 32. An expression cassette comprising a maize globulin promoter operably linked to a DNA segment which is complementary to a DNA molecule encoding a maize acetyl CoA carboxylase comprising an amino terminal chloroplast transit peptide.
 33. An expression cassette comprising a maize ubiquitin promoter operably linked to a DNA segment which is complementary to a DNA molecule encoding a maize acetyl CoA carboxylase comprising an amino terminal chloroplast transit peptide.
 34. The expression cassette of claim 32 or 33 wherein the DNA segment is complementary to a DNA molecule comprising SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22. 